RNA binding protein Pumilio2 promotes chemoresistance of pancreatic cancer via focal adhesion pathway and interacting with transcription factor EGR1

RNA binding protein Pumilio2 promotes chemoresistance of pancreatic cancer via focal adhesion pathway and interacting with transcription factor EGR1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article RNA binding protein Pumilio2 promotes chemoresistance of pancreatic cancer via focal adhesion pathway and interacting with transcription factor EGR1 Bangbo Zhao, Cheng Qin, Zeru Li, Yuanyang Wang, xiaoying Yang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5312328/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Feb, 2025 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted 5 You are reading this latest preprint version Abstract Background Pancreatic cancer (PC) has insidious onset, high malignancy and poor prognosis. Gemcitabine (GEM) is one of the first-line chemotherapy drugs for PC. However, resistance for GEM has always been a bottleneck problem leading to recurrence and death of PC patients. RNA-binding proteins (RBPs) are a kind of important proteins that regulate transportation, splicing, stability and translation of RNA. Abnormal expression of RBP often leads to a series of abnormal accumulation or degradation of downstream RNA resulting in various diseases. However, there is a lack of systematic study on whether RBPs play roles in GEM resistance of PC. Therefore, it is of great significance to explore RBPs and their specific molecular mechanisms that play an important role in GEM resistance of PC for further understanding and solving GEM resistance of PC. Methods RBPs closely related to GEM resistance of PC were screened based on transcriptome sequencing, siRNA library proliferation and GEM resistance test results. Relationship between expression level of PUM2 and clinicopathological variables was evaluated by immunohistochemical (IHC) staining of PC tissue chip. SRB proliferation assay, GEM drug resistance assay and transwell cell migration assay were used to detect the effects of PUM2 on the malignant biological behaviors of PC cells in vitro . Mice subcutaneous xenograft model was used to explore the effect of PUM2 in vivo . Furthermore, RIP-seq and RNA-seq were combined to explore the downstream mRNAs regulated by PUM2 in PC cells, and the regulation effect of PUM2 on downstream mRNAs was verified by qRT-PCR, Western Blot, RIP-qPCR, actinomycin D RNA stability assay, dual luciferase gene reporter assay and rescue experiments. Finally, transcription factors with mutual regulation relationship with PUM2 were screened by integrating data of RIP-seq, RNA-seq and JSAPAR database, and the regulatory relationship between the transcription factor EGR1 and PUM2 was verified by qRT-PCR, Western Blot, RIP-qPCR and rescue experiments. Results Several RBPs were found highly expressed in GEM resistant PC cell line. We screened out RNA-binding protein PUM2 as the most related RBP with GEM resistance of PC by siRNA library. IHC of PC tissue chip suggested that high expression of PUM2 was an independent risk factor for poor prognosis of PC patients. In vitro function experiments showed that PUM2 could promote proliferation, migration and resistance to GEM of PC cells. In vivo experiments showed that knockdown of PUM2 inhibited the growth of subcutaneous transplanted tumor in mice and increased sensitivity to GEM. Further, RNA-seq and RIP-seq were combined to explore the regulation role of PUM2 on downstream RNAs that promoted GEM resistance in PC. We found that PUM2 up-regulated mRNA stability of key genes (ITGA3, ADAM17, ASAP1, etc.) in the focal adhesion pathway. ITGA3 was verified to be the most significant downstream mRNA of PUM2 regulating GEM resistance in PC by rescue experiments in vitro , and PUM2 could stabilize ITGA3 mRNA by binding to PUM binding element (PBE) in the 3'UTR region of ITGA3 mRNA. Finally, we found the mutual regulation relationship between transcription factor EGR1 and PUM2, that is PUM2 binding to 3'UTR region of EGR1 mRNA, and EGR1 binding to promoter region of PUM2 gene, resulting in a cascade effect amplifying the role of PUM2 in PC chemoresistance. Conclusions RNA-binding protein PUM2 is closely related to the prognosis of PC patients. PUM2 promoted GEM resistance of PC by regulating mRNA stability of ITGA3 and other genes in focal adhesion pathway, and there was positive feedback regulation between PUM2 and transcription factor EGR1. The discovery of EGR1/PUM2/ITGA3 axis provided a solid experimental basis for the selection of chemotherapy regiments for PC patients and exploration of combined regimens to reverse GEM resistance in the future. Pancreatic cancer RNA binding protein Chemoresistance Gemcitabine PUM2 ITGA3 EGR1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background Pancreatic ductal adenocarcinoma (hereinafter referred to as pancreatic cancer, PC) has insidious onset, rapid progression and extremely poor prognosis. Up to now, the 5-year survival rate is still less than 10% [ 1 ]. Surgical resection is the only possible way to cure pancreatic cancer. However, due to the lack of clear clinical symptoms or signs in the early stage, 80–85% of pancreatic cancer patients have lost the chance of surgical resection when diagnosed [ 2 ]. For these patients with advanced stage or distant metastasis, chemotherapy is the most important treatment plan. The latest NCCN Clinical Practice Guideline for pancreatic cancer (version 2022.1) recommends that patients with pancreatic cancer receive chemotherapy regardless of pathological stage. Gemcitabine (GEM) based combination regimen is the most widely used first-line treatment regimen in clinical chemotherapy of pancreatic cancer at present [ 3 ]. GEM is a nucleoside antitumor drug that antagonizes nucleotide metabolism, and its derivatives can interfere with DNA synthesis and cell cycle to induce apoptosis [ 4 ]. However, GEM resistance in patients with pancreatic cancer is the biggest practical difficulty in clinical work at present. Taking postoperative adjuvant chemotherapy as an example, the proportion of GEM resistance within 3 years is as high as 78.6%. Even so, due to the poor efficacy or excessive toxicity of many other regimens, Gem-based chemotherapy is still the preferred chemotherapy regimen for most patients with pancreatic cancer [ 5 ]. Therefore, it is of great clinical significance to explore the mechanism of GEM resistance in pancreatic cancer to enhance the sensitization effect and reverse the drug resistance. The mechanism of GEM drug resistance is very complex, which is closely related to tumor microenvironment, instability of genetic material, regulation of intracellular signaling molecules, etc. Activation or inactivation of classical intracellular pathways is one of the main mechanisms causing primary and acquired drug resistance [ 6 ]. For example, inactivated p53 induces GEM drug resistance by activating the proliferation-promoting JAK2-STAT3 signaling pathway [ 7 ]. The downstream ARF6 protein of Kras/ERK signaling pathway can enhance the drug resistance of GEM by down-regulating the expression of dCK and hENT1 [ 8 ]. Gem-resistant pancreatic cancer cells can up-regulate the expression of tryptophan synthetase through MAPK signaling pathway and promote the synthesis of tryptophan to achieve metabolic reprogramming [ 9 ]. Mir-146a-5p can mediate GEM resistance of pancreatic cancer by activating NF-κB pathway [ 10 ]. At present, some therapeutic strategies have been used in combination with GEM to block the signaling pathway and reverse or delay the development of drug resistance, but most of them are ineffective. For example, Vismodegib plays an inhibitory role in cancer by inhibiting Hedgehog signaling pathway. In a phase II clinical trial, the combination regimen of Vismodegib and GEM did not significantly improve progression-free survival and overall survival of patients with metastatic pancreatic cancer compared with GEM alone (NCT01064622) [ 11 ]. Ibrutinib is a tyrosine kinase inhibitor. Although it has shown good safety in phase I/II clinical trials [ 12 ], in phase III clinical trials, Ibrutinib combined with chemotherapy still failed to show significant advantages in improving progression-free survival or overall survival (NCT02436668). In phase III clinical trials of Axitinib, a selective inhibitor of vascular endothelial growth factor receptor, GEM combined with Axitinib did not provide additional survival benefit for patients with advanced pancreatic cancer compared with monotherapy (NCT00471146) [ 13 ]. These failed clinical trials suggest that our understanding of the mechanism of GEM in pancreatic cancer is still limited, and the role of post-transcriptional regulation, post-translational modification and epigenetics in GEM resistance is still unknown. Therefore, starting from the above emerging gene molecular biological regulatory mechanisms, further study of GEM resistance mechanism and reversal of GEM resistance has a good prospect and hope. RNA Binding Protein (RBP) is a Protein that can bind to single or double stranded RNA molecules. RBPS usually contain at least one RNA binding domain (RBD) and are divided into different families according to the different RBDS contained. Currently known RBDS include: RNA recognition module (RBM), KH domain, double-stranded RNA binding domain (dsRBD), zinc finger protein domain (ZnF), PAZ domain, PIWI domain, SAM domain, etc. [ 14 ]. There are a large number of RBPS, accounting for about 6–8% of the protein coding genes in cells. At present, a total of 1171 eukaryotic RBPS have been recorded in RBP Database. RBPS bind to various Rnas and are widely involved in various stages of RNA molecular metabolism, which determines the fate of RNA from synthesis to decomposition. It plays an important biological role in cell development and cell metabolism. For example, mRNA splicing is the first step of RNA post-transcriptional processing, and the spliceosome that catalyzes this process is the RNA-protein complex formed by RBP. PolyA (polyadenylate) tail is an important component of eukaryotic mRNA, which plays an important role in mRNA nucleation, shearing, stability and translation [ 15 ]. The Cleavage and Polyadenylation Specificity Factor CPSF (RNA binding protein) is required in almost all mRNA tail adding processes. CPSF can bind to AAUAAA sequence and recruit and activate ployA polymerase activity together with ployA binding protein [ 14 ]. Another example is that mRNA is transferred from nucleus to cytoplasm after processing and maturation. TAP/NXF1 heterodimer is a key molecule mediating this process, but TAP cannot interact directly with RNA, so Aly/REF, an RNA-binding protein, is also required to participate. In addition, some RBPS (ZBP1, FMRP, etc.) can also mediate the intracellular localization of mRNA to achieve the specificity of the spatial localization of corresponding proteins [ 16 ]. In addition, mRNA translation in ribosomes is the last step of mRNA to produce functional proteins, and RBP is not only an important component of ribosomes, but also many RBPS regulate the translation of specific mrnas by recruiting or rejecting translation initiation complexes. Therefore, RBP is an important partner of mRNA, regulating all the key processes of mRNA molecule cleavage, editing, transport, degradation and translation, and is necessary for mRNA maturation and proper function. At the same time, RBP is also important for the generation and biological function of non-coding RNA. Therefore, RBP plays an indispensable role in a variety of life activities, and its abnormal expression or mutation will lead to the occurrence of a variety of diseases. At present, the biological function of RBP in the occurrence and development of pancreatic cancer is gradually being revealed, and many important findings and achievements have been made in the research of liver cancer, colorectal cancer and nervous system tumors [ 17 – 21 ]. Dozens of RBPS such as RNA-binding proteins HuR, CUGBP2, CPEB and IMP3 have been shown to regulate the proliferation, invasion and chemotherapy resistance of pancreatic cancer cells [ 22 ]. Taking HuR as an example, Costantino Tantamount first reported the high expression of HuR in pancreatic cancer cells in 2009, and the sensitivity of pancreatic cancer cells with high expression of HuR to GEM was significantly enhanced [ 23 ]. Jimbo et al. found that knockdown of HuR expression by shRNA could significantly reduce the invasion and metastasis ability of pancreatic cancer cells, whereas overexpression of HuR could enhance the invasion and metastasis ability of pancreatic cancer cells [ 24 ]. Lal et al. obtained HUR-null pancreatic cancer cell line by double knockout of HuR gene and found that the proliferation ability of HuR (-/-) cells was significantly decreased, and the tumorigenicity of HuR (-/-) pancreatic cancer cells in mice was also significantly decreased [ 25 ]. Other studies have shown that pancreatic cancer cells can survive and proliferate in the pancreatic cancer microenvironment of chronic hypoxia and nutrient deprivation by activating hypoxia inducible factors (HIFs) such as HuR [ 26 ]. Zarei et al. recently found that the regulatory axis of HuR-IDH1 is a key regulatory protein in the microenvironment of pancreatic cancer in the hypotrophic state, which can be used as a potential therapeutic target [ 27 ]. In addition, Blanco et al. demonstrated that HuR could promote the chemoresistance of pancreatic cancer cells in anaerobic environment by regulating the stability of PIM1mRNA, and the application of HuR inhibitor ms-444 could reverse the chemoresistance of tumor cells [ 28 ]. Li et al. demonstrated that HuR promoted the stability of IL-8 mRNA by regulating Mir-4312 translation and down-regulated the expression of BAG3 in pancreatic cancer cells, thus significantly reducing the migration and invasion ability of tumor cells [ 29 ]. However, even with these findings, we still know little about the function, molecular mechanism and expression regulation of many other RBPS in pancreatic cancer, especially GEM resistance in pancreatic cancer. Therefore, it is of great theoretical significance and clinical value to systematically identify the key RBPS involved in GEM resistance of pancreatic cancer, reveal the specific molecular mechanism involved in the occurrence of drug resistance, and develop new chemotherapy drug screening strategies and sensitization strategies for pancreatic cancer based on this target. Methods PDAC clinical specimens and cell lines Pancreatic ductal adenocarcinoma tissue was obtained from 82 PC patients who underwent operation in Peking Union Medical College Hospital and the pathological diagnoses were made by two pathologists independently. Clinical and pathological data were extracted from the medical record system of our center. Informed consents were obtained from each patient and the collection were approved by the Ethics Committee of Peking Union Medical College Hospital. PC cell lines used in our study were purchased from American Type Culture Collection (ATCC) except for AsPC-1/GEM, which was constructed by our research group previously. HPNE, MIA PaCa-2, PANC-1 and T3M4 were cultured in Dulbecco's Modified Eagle Medium (DMEM)/High Glucose (Hyclone). AsPC-1, ASPC-1/GEM and SW1990 were cultured in RPMI-1640 modified medium (Hyclone). BxPC-3 was cultured in RPMI (Corning). Capan-1 and CFPAC were cultured in Iscove's Modified Dubecco's Medium (IMDM, Hyclone). The culture medium of Capan-1 was added with 20% fetal bovine serum (FBS) and the culture medium of the other cell lines were added with 10% FBS. siRNA library screening Screening associated siRNA were designed and synthesized by RIBOBIO (Guangzhou, China). Three different siRNAs were designed targeting different sequences of a single gene, which were then mixed up before transfection. Reverse transfection were conducted in 96-well board using Lipofectamine TM 3000 (ThermoFisher, US). The transfection system was composed of 0.2μg siRNA mix, 0.2μL LipofectamineTM 3000 and 5000 cells. GEM was added after 24h of the seeding and the 96-well board was then cultured for another 48h before counting. Immunohistochemistry Immunohistochemical analyses were conducted according to standard procedures. Briefly, after deparaffinization, rehydration, antigen retrieval and endogenous peroxidase blockage, sections were incubated with anti-PUM2 antibody (1:100 dilution, Abcam, USA) at 4 °C overnight. Subsequently, sections were washed by PBS and incubated with HRP‐labeled secondary antibody for 30 min. After application of diaminobenzidine as a chromogen, the slides were evaluated using light microscopy (Olympus, Japan). PUM2 stable overexpression and knockdown cell lines construction To evaluate the function of PUM2, full-length human PUM2 cDNA was cloned into the pLentiGV492 expression vector (Genechem, Shanghai). For PUM2 knockdown assay, two short hairpin RNAs (shRNA) specifically targeting RALYL were cloned into the GV 493 lentiviral vector (Genechem, Shanghai). Stably transduced cells were selected by puromycin (Sigma-Aldrich). In vitro functional assays and animals Cells were seeded into 96-well plates for 8 hours before adding chemotherapeutic drugs. The concentration gradients of drugs were 0, 1nM, 10nM, 100nM, 1μM, 10μM, 100μM and 1M. After treating for 48 hours, drug-containing culture medium was replaced by fresh medium which contained 10% CCK-8 (Dojindo, Japan). Place the plates in incubator under 37℃ for 2 hours and determine its light absorption value at 450nm and 630nm using an enzyme-linked immunosorbent detector (Invitrogen, Thermo Fisher Scientific, USA). The difference between absorbance values at 450nm and 630nm indirectly reflects the number of living cells.Cell viability values under the drug concentration gradients were conducted non-linear fitting (inhibitor, four parameters) and IC50 was defined as the drug concentration where slope of the fit curve came to maximum (GraphPad Prism 9.0). A nude mouse xenograft model was used to evaluate tumor formation and chemoresistant ability in vivo. RNA extraction and quantitative real-time PCR (qRT-PCR) Total RNAs were extracted from cultured cell lines by Trizol reagent (Invitrogen, USA). Then cDNA was synthesized using the TaqMan Reverse Transcription Kit (Takara, Dalian, China) according to the manufacturer's instructions. For mRNA analysis, qRT-PCR was performed with SYBR® Premix Ex TaqTM Reagent (TaKaRa, Dalian, China) by using StepOne Plus Real-Time PCR system (Applied Biosystems, USA). Fold changes relative to β-actin were calculated using 2−ΔΔCt method. All primer sequences were listed in Supplementary Table 3. Western blot analysis Quantified protein lysates were resolved on SDS-PAGE, transferred onto a polyvinylidenedifluoride (PVDF) membrane (Millipore), and then blocked with 5% non-fat milk in Tris-buffered saline-Tween 20 (TBS-T) for 1 h at room temperature. The blocked membrane was then incubated with primary antibody diluted 1:1000 in 5% bovine serum albumin in TBS-T at 4 °C overnight. All antibodies used are listed in Supplementary Table 4. After washing with TBS-T, the membrane was incubated for 1 h with horseradish peroxidase HRP-conjugated secondary antibody. A complex of primary and secondary antibodies- labeled proteins were detected by enhanced chemiluminescence (ECL) system followed by exposure to 5200Multi visualizer (Tanon, China). RNA sequencing RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Total RNA was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase, then use RNaseH to degrade the RNA. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and dNTP. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing (Novogene Experimental Department) The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated. Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq2 R package (1.20.0). DESeq2 provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted P-value <0.05 found by DESeq2 were assigned as differentially expressed. Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the clusterProfiler R package, in which gene length bias wascorrected. GO terms with corrected Pvalue less than 0.05 were considered significantly enriched by differential expressed genes. KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-through put experimental technologies (http://www.genome.jp/kegg/). We used clusterProfiler R package to test the statistical enrichment of differential expression genes in KEGG pathways. The Reactome database brings together the various reactions and biological pathways of human model species. Reactome pathways with corrected Pvalue less than 0.05 were considered significantly enriched by differential expressed genes. The DO (Disease Ontology) database describes the function of human genes and diseases. DO pathways with corrected Pvalue less than 0.05 were considered significantly enriched by differential expressed genes. The DisGeNET database integrates human disease-related genes. DisGeNET pathways with corrected Pvalue less than 0.05 were considered significantly enriched by differential expressed genes. We used clusterProfiler software to test the statistical enrichment of differentially expressed genes in the Reactome pathway, the DO pathway, and the DisGeNET pathway. Gene Set Enrichment Analysis (GSEA) is a computational approach to determine if a pre- defined Gene Set can show a significant consistent difference between two biological states. The genes were ranked according to the degree of differential expression in the two samples, and then the predefined Gene Set were tested to see if they were enriched at the top or bottom of the list. Gene set enrichment analysis can include subtle expression changes. RNA immunoprecipitation (RIP) RNA immunoprecipitation (RIP) experiment was performed using Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) according to the manufacturer's instructions. Briefly, PC cells were lysed by RIP lysis buffer and then cell lysates were immunoprecipitated with protein A/G magnetic beads conjugated to anti-PUM2 anti-body (Abcam, USA) or normal rabbit IgG at 4 °C overnight. After RNA purification, qRT-PCR was used to measure the levels of focal adhesion pathway genes transcripts in the protein-RNA complexes. RIP sequencing Library Preparation and Quantification The library was constructed by Novogene Corporation (Beijing, China). Subsequently, pair-end sequencing of sample was performed on Illumina platform (Illumina, CA, USA). Library quality was assessed on the Agilent Bioanalyzer 2100 system. Data Analysis Raw data (raw reads) of fastq format were firstly processed using fastp (version 0.19.11, Chen et al., 2018) software. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low-quality reads from raw data. At the same time, Q20, Q30 and GC content of the clean data were calculated. All the downstream analyses were based on the clean data with high quality. Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using BWA (v 0.7.12) and clean reads were aligned to the reference genome using BWA mem (v 0.7.12). After mapping reads to the reference genome, we used the MACS2(version 2.1.0) peak calling software to identify regions of IP enrichment over background. A q-value threshold of 0.05 was used for all data sets. After peak calling, the distribution of chromosome distribution, peak width, fold enrichment, significant level and peak summit number per peak were all displayed. The interaction between transcript factor or chromatin histone modification and DNA were not random, while they show some specific sequence preference. Homer (Heinz et al., 2010) was used to detect the denovo sequence motif and the matched known motifs. Peak related genes can be confirmed by PeakAnnotator, and then Gene Ontology (GO) enrichment analysis of performed to identify the function enrichment results. GO enrichment analysis was implemented by the GOseq R package, in which gene length bias wascorrected. GO terms with corrected Pvalue less than 0.05 were considered significantly enriched by peak related genes. KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-through put experimental technologies (http://www.genome.jp/kegg/). We used KOBAS software to test the statistical enrichment of peak related genes in KEGG pathways. Different peak analysis was based on the fold enrichment of peaks of different experiments. A peak was determined as different peak when the odds ratio between two groups was more than 2. Using the same method, genes associated with different peaks were identified and conduct GO and KEGG enrichment analysis. RNA stability assay PUM2 stably knockdown and control MIA PaCa-2 and AsPC-1 cells were treated with Actinomycin D (Sigma-Aldrich) at 5 μg/mL. The time courses of samples with Actinomycin D treatment (0, 4, 8, 12 and 24 h) were used for RNA extraction. RNA was reversed transcription and analyzed by quantitative real-time PCR (qRT-PCR). Dual luciferase reporter assay GV272 luciferase expression system was purchased from Genechem Corporation (China) and performed according to the manufacturer’s instructions. ITGA3 reporter plasmid was cloned by inserting the full-length ITGA3-3′UTR and corresponding mutant sequence after the Firefly luciferase (F-luc) coding sequence. Cells seeded in 48-well plate were transfected with 100 ng of F-luc-ITGA3-3′UTR fusion reporter plasmid. After 72h, cells were analyzed with Dual Luciferase system (Yeason). F-luc activity was used to evaluate the binding effect of PUM2 on ITGA3-3′UTR. Renilla Luciferase (R-luc) was used to normalize the transfection efficiency of the reporter plasmid. Statistical analysis The GraphPad Prism version 9.0 (Graphpad, Inc., Chicago, IL) was used for data analysis. Patients’ survival rates were analyzed using Kaplan–Meier plots and log-rank tests. The correlations between PUM2 and different clinicopathological parameters were evaluated using Pearson’s χ2 test. Univariable and multivariable Cox proportional hazard regression models were used to analyze independent prognostic factors. Data are presented as the mean ± SD of three independent experiments. Results were considered statistically significant for p＜0.05. Results PUM2 plays a vital role in PDAC chemoresistance First, the sensitivity of AsPC-1/GEM, the GEM-resistant PC strain AsPC-1/GEM and the parental strain AsPC-1, to GEM previously constructed by our research group was verified. (IC50 = 21.67 μM) the sensitivity to GEM was significantly decreased (Fig. 1A). Transcriptome sequencing showed that there were 4551 differentially expressed genes between the GEM-resistant PC strain AsPC-1/GEM and the parental strain AsPC-1 (Fig. 1B). The differentially expressed genes were mainly enriched in the MAPK signaling pathway, tumor-associated glycoprotein tumors, Focal adhesion pathway, Hippo signaling pathway, Rap1 signaling pathway, arginine and proline metabolism pathway, pyrimidine metabolism and other pathways (Supplementary Fig. 1A), among them, the highly expressed genes in AsPC-1/GEM are mainly enriched in MAPK signaling pathway, Hippo signaling pathway, axon guidance pathway, tumor-related microRNA, tight junction, apoptosis, TGF-β pathway, platinum drug resistance, phosphoinositide metabolism, glutathione metabolism and other pathways (Supplementary Fig. 1B). Furthermore, we screened RBPs that may play a key role in the process of GEM resistance in PC through bioinformatics analysis and siRNA library (Fig. 1C). A total of 4551 genes with differential expression in the PC drug-resistant cell line AsPC-1/GEM and the parental strain AsPC-1 were crossed with 1183 RBPs to obtain the PC drug-resistant cell line AsPC-1/GEM and the parental strain There are 145 RBPs with differential expression in AsPC-1. Bioinformatics analysis based on the TGCA database and GTEx database was performed on the above 145 RBPs, and the candidate RBPs were screened by whether the gene had differential expression in PC tissue and normal pancreatic tissue. To 94 RBPs, the candidate RBPs were screened to 23 based on whether there was a significant difference in disease-free survival between patients with high and low expression of this gene (Supplementary Table 1). Then we used the si-RNA library of the above 23 RBPs for subsequent functional screening. First, we reverse transfected the siRNA library into MIA PaCa-2 cells and found that after knocking down RBPs such as PUM2, FLNA, RTN4, DUSP14, and EIF2AK2 for 72 h, the number of cells was significantly reduced compared with the control group (Fig. 1D-E). We selected 7 RBPs that may positively regulate cell proliferation ability for further drug resistance function screening test, and found that after knockdown of PUM2, the sensitivity of MIA PaCa-2 and AsPC-1 to GEM was significantly increased (Fig. 1F-I). In conclusion, among the differentially expressed RBPs (DE-RBPs) associated with PC GEM resistance in this screening, PUM2 is the RBP with the most significant effect on GEM sensitivity and proliferation ability of PC cells. We performed a separate bioinformatics analysis of PUM2 and found that it was highly expressed in PC tissues compared with normal pancreatic tissues (Fig. 1J, p < 0.05), and DFS was significantly shortened in PC patients with high PUM2 expression (Fig. 1K). In addition, we performed PUM2 immunohistochemical staining on PC tissue chips containing 82 samples, and scored each sample according to the staining intensity and staining area, and determined that 28 patients had high expression of PUM2 and 38 patients had moderate expression of PUM2 16 patients with low expression of PUM2, and then combined with the survival data of patients for survival analysis, it was found that the overall survival of PC patients with high expression of PUM2 was significantly shortened (Fig. 1L). Western Blot verified that the expression of PUM2 in the GEM-resistant PC strain AsPC-1/GEM was significantly higher than that in the parental strain AsPC-1 (Fig. 1M). We analyzed the correlation between the expression of PUM2 and the clinicopathological characteristics of the patients, and found that the expression of PUM2 had a strong correlation with the degree of tumor cell differentiation (p = 0.031) and T stage (p = 0.029) (Table 1). We defined the postoperative survival period greater than or equal to 24 months as long-term survival, and less than 24 months as short-term survival. Using univariate Cox regression analysis to explore the factors affecting the long-term survival of patients, we found that PUM2 was highly expressed (HR=16.671, 95CI% 4.882-56.922, p＜0.001), N1/2 stage (HR=2.797, 95CI% 1.263-6.197, p = 0.011) were risk factors affecting the long-term survival of patients, and the tumor was located in the body and tail of the pancreas (HR=0.409, 95CI% 0.174-0.964, p = 0.041) is a protective factor affecting the long-term survival of patients. Further multivariate Cox regression analysis was used to explore the factors affecting the long-term survival of patients. It was found that PUM2 was highly expressed (HR=15.280, 95CI% 4.461-52.336, p<0.001) It is an independent risk factor affecting the long-term survival of patients (Table 2). PUM2 promotes proliferation, migration and chemoresistance of PDAC Next, we performed functional experiments to verify the effect of PUM2 on the malignant biological behavior of PC cells. Using Western Blot and qRT-PCR, we obtained the expression profile of PUM2 in commonly used PC cell lines at the protein and RNA (Fig. 2A-B) levels and found that PUM2 was relatively high in MIA PaCa-2, AsPC-1 Expression, relatively low expression in BxPC-3, PANC-1. We used lentivirus to construct cell lines with stable knockdown of PUM2 in MIA PaCa-2 and AsPC-1, and constructed cell lines with stable overexpression of PUM2 in BxPC-3 and PANC-1, and performed Western Blot and qRT-PCR. The results showed that the expression level of PUM2 in the two PUM2 stable knockdown cell lines of MIA PaCa-2 PUM2 KD and AsPC-1 PUM2 KD was significantly lower than that of the control cell line (Fig. 2C-D), while BxPC-3 PUM2 OE, PANC The expression level of PUM2 was significantly higher in the two PUM2-overexpressing cell lines -1 PUM2 OE than in the control cell line (Fig. 2E-F). Using the PC PUM2 stable knockdown and overexpression cell lines constructed above, we performed in vitro functional tests. The results of proliferation experiments showed that knockdown of PUM2 could significantly inhibit the in vitro proliferation of PC cells MIA PaCa-2 and AsPC-1 (Fig. 2J-H), overexpression of PUM2 can promote the in vitro proliferation ability of significant PC cells BxPC-3 and PANC-1 (Fig. 2I-J). GEM cytotoxicity assay showed that knockdown of PUM2 could increase the sensitivity of PC cells MIA PaCa-2 and AsPC-1 to GEM (Fig. 2K-L), while overexpression of PUM2 could reduce the sensitivity of PC cells to GEM (Fig. 2M-N). Transwell cell migration experiments showed that knockdown of PUM2 could significantly inhibit the migration ability of PC cells (Fig. 2O-P, p < 0.001), and overexpression of PUM2 could significantly promote the migration ability of PC cells BxPC-3 and PANC-1 (Fig. 2Q-R, p < 0.001). Furthermore, we used a mouse subcutaneous xenograft tumor model to verify the effect of PUM2 on PC cell proliferation and GEM sensitivity in vivo (Fig. 3A). We subcutaneously injected two groups of MIA PaCa-2 PUM2 NC cells and MIA PaCa-2 PUM2 KD cells into nude mice, respectively. After 2 weeks of injection, clear tumor formation was observed. GEM (50 mg/kg) was injected intraperitoneally for the second time, and PBS was injected in the other two groups. After 4 weeks of continuous administration, the mice were sacrificed, the transplanted tumors were dissected out and the tumor and volume were measured, and the tumor growth curve was made. The results showed that the PUM2 NC+PBS group had the fastest tumor growth rate, followed by the PUM2 KD+PBS group, while the tumor growth rate was the slowest in the PUM2 KD+GEM group (Fig. 3B). During the 45-day tumorigenic cycle, the subcutaneous tumors of the mice in the PUM2 KD+GEM group had almost no obvious growth, and finally the subcutaneous tumor weight of the mice in this group was significantly lower than that of the mice in the other groups (Fig. 3C-D, p<0.001). RNA-seq and RIP-seq jointly reveals RNA regulating function of PUM2 in PDAC Next, we explored the possible mechanisms by which PUM2 exerts the above-mentioned biological functions in PC, especially in promoting resistance to GEM. First, we performed transcriptome sequencing on the MIA PaCa-2 PUM2 KD stable transfected cell line and its control cell line, MIA PaCa-2 PUM2 NC. Three samples of KD and PUM2 NC can be clustered separately, with small differences within groups and large differences between groups (Supplementary Fig. 2A-B). The sequencing results showed that after knockdown of PUM2, the expression of 2607 genes were significantly up-regulated, the expression of 1966 genes were significantly down-regulated, and the expression of the remaining 22,701 genes had no significant difference between the two groups (Fig. 4A, Supplementary Fig. 2C). Furthermore, we performed GO and KEGG enrichment analysis on the differentially expressed genes after knockdown of PUM2 to find the important pathways and cell biological functions that PUM2 may regulate. In GO enrichment analysis, all differentially expressed genes can be enriched for cell adhesion molecule binding, cell adhesion junction, focal adhesion pathway, chromatin binding, ubiquitin protein ligase binding, cell cycle negative regulation, RNA metabolism process, Important pathways such as ribosome assembly, the analysis of genes whose expression is up-regulated after PUM2 knockdown can be enriched to important pathways such as NADH deoxygenase activity, oxidoreductase activity, mitochondrial protein complex, and ribosomal protein complex biosynthesis. Analysis of genes down-regulated after PUM2 enriched important pathways such as serine/threonine kinase activity, centrosome, proteasome catalytic process, transcriptional repression complex, and regulation of cell-matrix junctions (Fig. 4B, Supplementary Fig. 2D-E). In the KEGG enrichment analysis, the analysis of all differentially expressed genes can enrich important pathways such as endocrine resistance, longevity regulation pathways, tumor glycoproteins, oxidative phosphorylation, apoptosis, and cell cycle. Gene analysis can enrich important pathways such as N-glycan biosynthesis, cell cycle, spliceosome, oxidative phosphorylation, and thermogenesis. The analysis of genes whose expression is down-regulated after PUM2 knockdown can be enriched in the focal adhesion pathway, NF -κB signaling pathway, mitophagy, PI3K-AKT signaling pathway, tumor-related choline metabolism, ErbB signaling pathway, longevity regulation pathway, TNF signaling pathway, FoxO signaling pathway, PC-related genes and other important pathways (Supplementary Fig. 3A-C). In addition, GSEA analysis also showed that knockdown of PUM2 significantly decreased the expression of related genes in the focal adhesion pathway in PC cells (Fig. 4C). Further, we performed RNA co-immunoprecipitation (RIP) experiments of PUM2 protein on MIA PaCa-2 cells, and the RNA products from RIP were exported for RIP-seq. The measured peaks were distributed on all 23 pairs of chromosomes including sex chromosomes (Supplementary Fig. 4A). By matching each peak to the corresponding gene region, it was found that most of the peaks were located in the 3'UTR region and CDS region of the gene (Supplementary Fig. 4B), of which 45.49% of the peaks were matched to the 3'UTR region of the corresponding gene in the genome, and 44.62% of them were matched to the 3'UTR region of the corresponding gene in the genome. The peaks of PUM2 were matched to the CDS regions of the corresponding genes in the genome, and the above two accounted for more than 90% of all peaks (Supplementary Fig. 4C), which was also in line with the previous conclusion that the RNA-binding element PBE of the PUM2 protein mainly binds to the 3'UTR of mRNA. In addition, the enrichment analysis of the genes corresponding to the peaks measured in RIP-seq showed that the focal adhesion pathway, Wnt signaling pathway, Ras signaling pathway, tumor-associated glycoprotein, PI3K-AKT signaling pathway, Notch signaling pathway, mTOR signaling pathway and other important Pathway gene sets were enriched (Fig. 4D). Analyzing the results of RNA-seq detection, there were many key pathways related to GEM resistance in previous studies in the KEGG enrichment analysis of down-regulated genes after PUM2 knockdown. Therefore, we compared the peaks obtained in RIP-seq with RNA- The genes whose expression was down-regulated after PUM2 knockdown obtained in seq were intersected, and a total of 267 genes were obtained (Fig. 4E). The enrichment analysis of these genes also obtained cell adhesion, chromatin binding, transcriptional repression complex, and transcription factor binding. and other important pathways (Supplementary Fig. 4D). Because focal adhesions and cell adhesion pathways appear in the GO enrichment analysis, KEGG enrichment analysis, RIP-seq peak corresponding gene enrichment analysis and the enrichment analysis results of the two intersection genes of RNA-seq, and the gene set contains ITGA3 gene, that is, the gene with the most obvious difference between the PUM2 knockdown group and the control group in RNA-seq, and previous literature also showed that ITGA3 plays an important role in GEM resistance. Therefore, we selected ITGA3 and its focal adhesion pathway gene set as the possible downstream of PUM2 in regulating GEM resistance in PC for further study (Supplementary Table 2). The abundance of corresponding RNA of typical genes in focal adhesion pathway was significantly higher in PUM2-Ab than that in input (Fig. 4F). We performed bioinformatics analysis based on TCGA and GTEx databases for six candidate genes including ITGA3, ADAM17, ASAP1, FLNA, COL1A2, and COL6A3. The results showed that the above genes were highly expressed in PC tissues, and their expression was significantly positively correlated with the expression level of PUM2. For some genes in the focal adhesion pathway gene set including the above genes (9/27), there was a significant difference in OS between high expression PC patients and low expression patients (p＜0.05); for focal adhesion pathways including the above genes For half of the genes in the pathway gene set (13/27), there was a significant difference in DFS between patients with high expression and low expression (p＜0.05) (Supplementary Table 2, Supplementary Fig. 5). Focal adhesion pathway is regulated integrally by PUM2 at mRNA level in PDAC In order to explore whether PUM2 has a regulatory effect on genes related to the focal adhesion pathway gene set such as ITGA3, and the specific mechanism of PUM2 regulating the above genes, we carried out the following experiments to verify. First, we used qRT-PCR to detect the mRNA levels of representative genes (ITGA3, ADAM17, ASAP1, FLNA, COL1A2, COL6A3) in the focal adhesion pathway in the combined analysis of RIP-seq and RNA-seq, and found that knockdown of PUM2 After overexpression of PUM2, the mRNA levels of the above genes in MIA PaCa-2 and AsPC-1 cells were significantly decreased (Fig. 5A-B), and the mRNA levels of the above genes in MIA PaCa-2 and AsPC-1 cells were significantly increased after PUM2 overexpression (Fig. 5C-D), the results confirmed that the mRNA levels of ITGA3, ADAM17, ASAP1, FLNA, COL1A2, and COL6A3 were indeed correlated with the expression levels of PUM2. Secondly, we used RIP-qPCR to verify that PUM2 can bind to the 3'UTR region of the above gene mRNAs, and found that compared with IgG, the PUM2 antibody group could significantly bind to the 3'UTR regions of ITGA3, ADAM17, ASAP1, FLNA, COL1A2 and COL6A3 gene mRNAs (Fig. 5E-H). PUM2 binds to the 3'UTR region of ITGA3 mRNA at the highest level. Subsequently, we used ActD to conduct the RNA stability test of the above genes, and found that after knockdown of PUM2 in both MIA PaCa-2 and AsPC-1 cells, ITGA3, ADAM17, ASAP1, FLNA, COL1A2 and COL6A3 The stability of isogenic mRNA decreased to varying degrees, confirming that PUM2 regulates the mRNA stability of the above-mentioned genes by binding to the 3'UTR region of the above-mentioned genes, and finally regulates the mRNA levels of the above-mentioned genes (Fig. 5I-J). ITGA3 is a crucial downstream in PUM2 promoting chemoresistance and metastasis of PDAC ITGA3 is the gene with the most significant difference between the PUM2 knockdown group and the control group in RNA-seq, and its mRNA 3'UTR region is also the most enriched in RIP-qPCR. In addition, a study published in Gastroenterology in 2020 It is shown that ITGA3 can be activated and up-regulated by the upstream ZIP4/ZEB1 pathway in PC cells, thereby activating the downstream JNK pathway and inhibiting the GEM transporter ENT1 on the cell membrane, thereby inducing PC GEM resistance. Therefore, we validated ITGA3 as a key downstream gene of PUM2 to induce GEM resistance in PC cells. First, we performed a dual-luciferase reporter assay to verify the binding of PUM2 to the ITGA3 3'UTR region. By searching the sequence of the ITGA3 3'UTR region, we found that it contains the PBE sequence (-TGTATATA-, Fig. 6A). Based on this, we constructed a wild-type firefly luciferase reporter gene plasmid and a PBE sequence in the 3'UTR region of ITGA3 mRNA, respectively. The PBE sequence mutant firefly luciferase reporter gene plasmid in the 3'UTR region of ITGA3 mRNA was used for the dual luciferase reporter gene assay. The results showed that after knocking down PUM2, the PBE sequence in the 3'UTR region of ITGA3 mRNA was expressed in the middle and downstream of the wild-type plasmid. The degree of expression was significantly reduced (p < 0.01), while the expression degree of the downstream genes of the PBE sequence mutant plasmid in the 3'UTR region of ITGA3 mRNA had no significant change (Fig. 6B). The above experiments proved that PUM2 can bind to the PBE sequence in the 3'UTR region of ITGA3 mRNA. Subsequently, we used Western Blot to verify that after knockdown of PUM2, the protein levels of ITGA3 in MIA PaCa-2 and AsPC-1 cells were significantly decreased, and the protein levels of key proteins AKT and p-AKT in the AKT pathway downstream of the focal adhesion pathway also decreased. significantly decreased (Fig. 6C), while the protein levels of ITGA3, AKT, and p-AKT were significantly increased in BxPC-3 and PANC-1 cells after overexpression of PUM2 (Fig. 6D). Finally, we performed rescue experiments for the function of PUM2 using ITGA3. ITGA3 was overexpressed in MIA PaCa-2 and AsPC-1 cells stably knocked down by PUM2 and verified by Western Blot. The results showed that the cell model was successfully constructed (Fig. 6E). Furthermore, we examined MIA PaCa-2 PUM2 NC cells, MIA PaCa-2 PUM2 KD cells, MIA PaCa-2 PUM2 NC+Vec cells, MIA PaCa-2 PUM2 KD+ITGA3 OE cells and AsPC-1 PUM2 NC cells, AsPC-1 PUM2 KD cells, AsPC-1 PUM2 NC+Vec cells, AsPC-1 PUM2 KD+ITGA3 OE cells, a total of 8 cell lines were sensitive to GEM. It was found that overexpression of ITGA3 could reverse the effect of knockdown of PUM2 in the above cell lines. The effect of increased sensitivity to GEM (Fig. 6F-G). In addition, because the focal adhesion pathway genes including ITGA3 also play an important role in cell adhesion, invasion and metastasis, we tested the migration ability of the above 8 cell lines. The results showed that overexpression of ITGA3 could also reverse the cells after knockdown of PUM2. The effect of reduced mobility (Fig. 6H-I, p<0.001). EGR1 and PUM2 regulate mutually in PDAC resulting in a cascaded effect After the preliminary exploration of the key genes downstream of PUM2 was completed, we turned to further explore the upstream transcription factors that regulate the expression of PUM2. We used the JASPAR database to predict the transcription factors that can bind to the promoter region of PUM2, and then analyzed the predicted possible upstream transcription factors of PUM2 (366), the corresponding genes of mRNAs that could bind to PUM2 in RIP-seq (1535) and RNA- In seq, the down-regulated genes (1966) were crossed after PUM2 knockdown (Fig. 7A), and finally 22 transcription factors were obtained. Bioinformatics analysis showed that among the 22 transcription factors, early growth factor 1 (EGR1) was significantly higher in PC tissues than in normal PC tissues (Figure 7B, p < 0.05), and the expression level of EGR1 in PC tissues It was significantly positively correlated with the expression level of PUM2 (Fig. 7C, R = 0.29, p = 9.7×10-5). Then reverse verification was performed, using the CLIP-seq or RIP-seq data in the PARalyzer data set, the FIMO data set and the ENCODE eCLIP data set in the POSTAR website to predict the RNA-binding proteins that may bind to EGR1 mRNA. The results are shown in multiple data sets. Concentratingly, both PUM2 may bind to EGR1 mRNA (Fig. 7D). In addition, a study published in Science in 2020 showed that EGR1 plays an important role in pancreatitis-cancer transformation. Therefore, based on the forward and backward prediction results, bioinformatics analysis results and literature reading, we finally selected EGR1 as a candidate upstream transcription factor of PUM2 for follow-up research. Furthermore, we verified the expression regulation relationship between transcription factor EGR1 and RNA-binding protein PUM2 at the RNA and protein levels. The results showed that after knockdown of PUM2 in MIA PaCa-2 and AsPC-1 cells, the expression of EGR1 at both mRNA and protein levels was significantly decreased (Fig. 8A-B), and PUM2 was overexpressed in BxPC-3 and PANC-1 cells After treatment, the expression of EGR1 at both mRNA and protein levels was significantly increased (Fig. 8C-D). Conversely, after knockdown of EGR1 in BxPC-3 cells, the expression of PUM2 at the mRNA and protein levels was also significantly decreased (Fig. 8E-F). After overexpression of EGR1 in PANC-1 cells, the expression of PUM2 at the mRNA and protein levels was significantly reduced Expression was also significantly elevated (Fig. 8G-H). In terms of the regulatory mechanism of the two, we confirmed by RIP-qPCR that in MIA PaCa-2 and AsPC-1 cells, PUM2 protein can more significantly bind to the 3'UTR region of EGR1 mRNA than IgG (Fig. 8I-J, p＜0.01). Finally, we performed rescue experiments for the function of PUM2 using EGR1. EGR1 was overexpressed in MIA PaCa-2 cells stably knocked down by PUM2 and verified by Western Blot, and the results showed that the cell model was successfully constructed (Fig. 8K). Furthermore, we detected the sensitivity of four cell lines to GEM, including MIA PaCa-2 PUM2 NC cells, MIA PaCa-2 PUM2 KD cells, MIA PaCa-2 PUM2 NC+Vec cells, and MIA PaCa-2 PUM2 KD+EGR1 OE cells. It was found that overexpression of EGR1 could reverse the effect of increased GEM sensitivity caused by knockdown of PUM2 in the above cell lines (Fig. 8L), and overexpression of EGR1 could also reverse the effect of decreased cell migration ability after knockdown of PUM2 (Fig. 8M, p＜0.001). Discussion PC has a high degree of malignancy. Chemotherapy is one of the most important treatment methods for PC. Effective chemotherapy can not only prolong the survival of advanced patients, but also transform unresectable or junctional resectable patients into resectable ones, thereby enabling patients to have a cure. possible. Therefore, how to sensitize PC chemotherapy efficacy and reduce the occurrence of PC chemotherapy resistance has always been one of the key contents in the basic research of PC. At present, the first-line chemotherapy regimens for PC are mainly divided into three categories. One is a combination chemotherapy regimen based on GEM (such as GEM combined with nab-paclitaxel), and the other is based on 5-FU or its derivatives. Combination chemotherapy regimens (such as FOLFIRINOX regimen), and the third is platinum-containing regimens (such as FOLFOX regimen) applied to patients with BRCA mutations. Currently, clinical practice in China mostly adopts the first chemotherapy regimen, that is, GEM-based combination chemotherapy regimen. Therefore, in-depth study of the GEM resistance mechanism of PC and the search for targeted therapy to sensitize GEM are effective means to improve the efficacy of PC chemotherapy. RBP is an essential protein in the process of intracellular RNA metabolism. RNA is a key part of the central dogma. Therefore, RBP that regulates its various metabolic processes is also of great significance to the regulation of cellular functions. Although some studies on RBP in PC have been reported, there is still a lack of systematic research on which RBP plays a key role in GEM resistance of PC. Therefore, this subject has carried out in-depth research from exploration to verification, from in vivo to in vitro, and from function to mechanism, with the scientific question of exploring the functions and specific mechanisms of key RBPs in GEM resistance of PC. Based on the transcriptome sequencing data of GEM-resistant PC strains and parental strains, and PC gene expression and survival data from the TCGA database, we first screened out candidate RBPs that may be related to GEM resistance in PC, and then constructed siRNA for proliferation and GEM The cytotoxicity test showed that PUM2 was the RBP with the strongest correlation with GEM resistance in PC. Based on the analysis of PUM2 immunohistochemical staining and clinicopathological characteristics of PC tissue chips, it was confirmed that the high expression of PUM2 is closely related to the poor prognosis of PC. PUM2, one of the mammalian Pumilio proteins, is a member of the PUF family of sequence-specific RNA-binding proteins [ 30 ]. Pumilio protein was first discovered because of its important role in Drosophila embryonic development [ 31 ] and has since been widely regarded as a typical post-transcriptional regulator. There are two kinds of Pumilio proteins in the human body, namely PUM2 and PUM1, which have 76% homology and 30% homology with the Pumilio protein in Drosophila. The structural feature of PUM2 is that it contains 1 interspecies homology. The highly conserved Pumilio homology domain (PUM-HD) [ 32 , 33 ] also contains a conserved sequence that can bind to mRNA, the Pumilio recognition element (PRE) [ 34 , 35 ]. In recent years, the function of PUM2 in tumorigenesis and development has been gradually recognized [ 36 ]. For example, PUM2 is highly expressed in acute myeloid leukemia tumor cells, which can affect the proliferation, cell cycle regulation and apoptosis of hematopoietic stem cells. In terms of mechanism, PUM2 can use PRE to bind to FOXP1 mRNA to activate FOXP1 expression, and ultimately inhibit cell cycle inhibitory factors, for example, the expression of CDKN2B [ 37 ]; another example, PUM1 in the PUM family in seminoma functions as a post-transcriptional repressor, which can inhibit the expression of SPIN1, an important regulatory protein of meiosis at the post-transcriptional level, thereby inhibiting spermatogenesis. Proliferation of primary cell tumors and promote apoptosis [ 38 ]. In addition to mRNA, PUM proteins can also affect the occurrence and development of tumors by regulating the functions of non-coding RNAs such as miRNA and lncRNA [ 39 ]. For example, in glioblastoma, CDKN1B mRNA can be bound by miR-221 and miR-222, and PUM2 can cause local conformational rearrangement of CDKN1B mRNA by binding to the 3'UTR region, which in turn leads to the exposure of complementary miRNA binding sites and synergy Enhance the inhibitory effect of miRNA on CDKN1B expression [ 40 , 41 ]. In this study, to explore the mechanism of PUM2 promoting PC resistance to GEM, we combined RIP-seq and RNA-seq to find that PUM2 can bind to the 3'UTR region of focal adhesion-related gene mRNAs such as ITGA3, thereby improving mRNA stability The expression levels of focal adhesion-related genes were up-regulated, and the regulatory effect of PUM2 on ITGA3 was verified step by step by RIP-qPCR, RNA stability experiments, dual luciferase reporter experiments and functional rescue experiments. The post-transcriptional regulation of PUM2 on downstream target mRNAs is mostly post-transcriptional inhibition [ 42 ]. The 5'7-methylated guanylate cap (5'cap) and 3 The 'polyadenylation tail is the main structure that prevents mRNA from being degraded [ 43 ], while PUM2 binds to the 3' polyadenylation tail of mRNA and recruits the CNOT family with deadenylase to induce mRNA degradation[ 43 ] 44, 45]. In addition, the N-terminus of PUM2 also has a conserved repression domain (RD), which is currently believed to bind to the 5' cap structure of mRNA to produce a "decapping" effect to accelerate mRNA degradation [ 46 ]. At the same time, the Pum-HD structure in PUM2 can inhibit the role of PABP in promoting transcription initiation without dissociating PABP protein from mRNA, but the specific mechanism is currently unknown [ 47 ]. In addition to post-transcriptional inhibition, PUM2 can also play a post-transcriptional activation effect on some genes. For example, in the aforementioned acute myeloid leukemia, PUM2 can play a post-transcriptional activation role by binding to the 3'UTR region of FOXP1 mRNA and upregulate its expression. [ 37 ]. Therefore, PUM2 plays different post-transcriptional regulatory roles for different genes. Similarly, iron response element protein (IREP) plays different post-transcriptional regulatory roles depending on the sequence of the binding site. Cytoplasmic poly-adenylation element-binding protein (CPEB) can also exert different post-transcriptional regulatory roles according to different developmental cues [ 48 , 49 ]. This study confirms that PUM2 can up-regulate the expression level of focal adhesion-related genes by inhibiting the degradation rate of focal adhesion-related genes in PC cells, but the specific mechanism of the deeper PUM2-mediated post-transcriptional activation has not been reported in previous studies. In the follow-up study, we can conduct Co-IP experiment and RNA pull down experiment on PUM2 protein in PC, and even try to conduct Co-IP experiment of PUM2 protein on the protein product of RNA pull down experiment, to explore whether PUM2 can interact with PUM2. Other post-transcriptional regulators, such as proteins that promote 3' polyadenylation, form a complex to play a role in post-transcriptional activation; or whether PUM2 competes with important post-transcriptional repression regulators of focal adhesion-related gene mRNAs Inhibition and post-transcriptional activation. The key downstream gene of PUM2 in this study is ITGA3, which encodes the α3 subunit of the integrin family. Integrin proteins are a class of ubiquitous heterodimeric transmembrane glycoprotein receptors, mainly present on mammalian cell membranes as signal transduction proteins [ 50 ]. Previous studies have shown that the integrin family can promote PC metastasis and resistance to chemotherapy. For example, integrin α2β1 increases the resistance of PC cells to 5-FU by upregulating the expression of Bcl-2 [ 51 ]; another example, ITGB1 can promote GEM resistance of PC by activating Cdc42 [ 52 ]. The integrin α3 subunit often non-covalently binds to the β1 subunit to form the integrin α3β1 receptor anchored on the cell membrane. A study published in "Gastroenterology" in 2020 showed that the zinc finger protein ZIP4 promotes the transcription factor ZEB1 by activating the STAT pathway The expression of integrin α3β1 on the cell membrane can then activate the intracellular JNK pathway, thereby inhibiting the expression of the GEM transporter ENT1 on the cell membrane, and finally promoting the GEM resistance of PC cells function [ 53 ]. Therefore, the up-regulation of ITGA3 expression caused by the high expression of PUM2 in this study can be clearly regarded as one of the factors explaining the GEM resistance of PC induced by PUM2. In recent years, many drugs targeting the integrin family have appeared. Although there is still no FDA-approved integrin family-targeting drug for tumor therapy, some of them have been used in tumor-related clinical trials [ 54 ]. For example, GLPG-187, which targets integrins containing the αv subunit, is being tested for the treatment of various solid tumors, and Cilengitide, which targets integrins αvβ3 and αvβ5, is being tested for the treatment of glioblastoma, OS2966, which targets β1 subunit-containing integrins, was tested for the treatment of glioma [ 54 ]. At present, it may be difficult to develop small molecule inhibitors or monoclonal antibodies targeting PUM2 from scratch. Instead, we can try to inhibit PUM2 to play an important downstream ITGA3 in promoting GEM resistance in PC to achieve GEM sensitization. There is also no specific drug targeting integrins containing α3 subunit, but in the follow-up experiments, it can be considered to use OS2966 targeting integrins containing β1 subunit to inhibit the function of integrin α3β1 and achieve GEM sensitization effect. In addition, combined with the rapid development of artificial intelligence and deep learning technology this year, we will consider applying deep learning methods in the future to find drugs that may inhibit PUM2 or ITGA3 among FDA-approved drugs, that is, "Drugs targeting PUM2 or ITGA3" repropose”, which will be subsequently verified by cell and animal experiments, in order to transform this topic into an effective study that can truly sensitize PC GEM chemotherapy. In addition to the focal adhesion-related gene set including ITGA3, many pathways were enriched in RIP-seq and RNA-seq in this study, and these pathways may also be involved in PUM2-induced PC GEM resistance, and even PUM2-induced PC It plays an important role in other malignant biological behaviors of cancer. For example, NF-κB signaling pathway, FoxO signaling pathway enriched in RNA-seq, Wnt signaling pathway, Notch signaling pathway, and mTOR signaling pathway enriched in RIP-seq have all been reported to be related to the occurrence and development of PC, and even PC GEM resistance is closely related [ 55 – 59 ]. It is worth noting that in the two functional enrichment analysis of RNA-seq in this study and the gene enrichment results of up-regulated expression after PUM2 knockdown, oxidative phosphorylation-related gene sets, NADH activity-related gene sets, and oxidoreductases appeared. Activity-related gene set, located in mitochondrial inner membrane protein gene set. Specific to specific genes, after knocking down PUM2 in this study, mitochondrial ribosomal proteins (mitochondrial ribosomal proteins, MRPs), isocitrate dehydrogenase (IDH2, IDH3G), mitochondrial membrane proteins (VDAC1, TOMM40, CHCHD1, CHCHD2, CHCHD10 etc.) expression was significantly increased, suggesting that after knockdown of PUM2, the aerobic respiration efficiency of PC cells may increase, and it can be speculated that the aerobic respiration efficiency is low when PUM2 is highly expressed, and the energy supply of cells may mainly depend on anaerobic glycolysis. This is consistent with the conclusion of previous studies that anaerobic glycolysis can promote GEM resistance in PC [ 60 ]. By reading the literature, we were pleasantly surprised to find that the phenomenon that PUM2 promotes anaerobic glycolysis is not our speculation. A study published in "Molecular Cell" in 2019 suggests that PUM2 is an RBP that plays an important role in the regulation of aging. The high expression of PUM2 in aging cells can downregulate the expression of its downstream mitochondrial fission factor (Mff). Decreased fission ability leads to mitochondrial dysfunction, whereas knockdown of PUM2 can enhance mitochondrial function. The Seahorse results in this paper also confirmed that in the case of knockdown of PUM2, the rate of cellular aerobic respiration was significantly reduced [ 61 ]. Therefore, we intend to focus on mitochondrial function and oxidative respiration as an entry point to study whether PUM2 plays a more important and extensive biological function in PC cells by regulating mitochondrial function and leading to cellular metabolic reprogramming. It should be noted that only taking the perspective of PUM2 regulating the downstream mRNA metabolism level as an entry point may still not fully explain and solve the major problem of GEM resistance in PC. Key cues for regulatory function. In 2016, a study published by S. Lee et al. in "Cell" showed that noncoding RNA activated by DNA damage (NORAD), which is closely related to DNA damage, contains 15 PBE sequences, and the high expression of NORAD can be expressed in the cytoplasm. The local enrichment of PUM2 in the interior leads to the phenomenon of phase separation, which promotes DNA damage repair and the expression level of cell cycle-related genes, while the inactivation of NORAD leads to a decrease in the level of NORAD-PUM2 phase separation, which can eventually lead to intracellular staining through chromosomal abnormalities and mitotic abnormalities. It is qualitatively unstable, which is of great significance for tumorigenesis [ 62 ]. Although the group's follow-up study suggested that NORAD is more distributed in the nucleus under stress, and that the RNA-binding protein RBMX rather than PUM2 plays a role in NORAD-mediated regulation of chromatin stability, other studies have suggested the important role of PUM2 in this process [ 63 ]. Two studies published in eLife confirmed from in vitro and in vivo conditions that NORAD in the cytoplasm can function as a PUM2 sponge independent of stress state, thereby regulating genome stability and mitotic processes, and PUM2 overexpression can simulate A phenotype of genomic instability and mitochondrial dysfunction resulting from NORAD deletion [ 64 , 65 ]. Taken together, the above studies are not only in line with the high expression of PUM2 may lead to mitochondrial dysfunction, but also suggest that high expression of PUM2 may lead to serious consequences of intracellular genome instability and mitotic dysregulation. Therefore, in follow-up studies, we will also focus on whether the expression level of PUM2 in PC cells is related to genome stability, and whether PUM2 may act as a key driver gene to cause PC through the chromatin instability pathway. In the last part of this study, we also explored the transcription factors that may interact with PUM2, and found that PUM2 and EGR1 can function as RNA-binding proteins and transcription factors, respectively, to promote the expression of each other. Therefore, the two are in PC. A cascade amplification effect is formed in the occurrence of GEM resistance. In this study, the RIP-qPCR experiment was used to verify the direct binding effect of PUM2 on EGR1 mRNA, and the ChIP-qPCR experiment can be used to further verify the binding effect of EGR1 and the PUM2 gene promoter region. EGR1, a member of the early growth response factor (EGR) family, can bind to the EGR1-binding sequence (EBS) in the promoter region of downstream genes, CC(A/T)6GG, Binding to promote the expression of downstream genes [ 66 , 67 ]. Growth factors, tumor necrosis factors, inflammatory factors, radiotherapy, and reactive oxygen species can induce increased expression of EGR1 [ 68 , 69 ]. EGR1 is highly expressed in gliomas, lung cancers, gastrointestinal tumors, and melanomas. EGR1 can play a role in promoting tumor cell proliferation, invasion and metastasis [ 70 – 73 ]. For example, EGR1, whose expression is up-regulated by the activated MAPK pathway in prostate cancer cells, can directly bind to the promoter region of the Cyclin D1 gene, thereby up-regulating the expression of Cyclin D1 and promoting the proliferation of prostate cancer cells [ 74 ]; for another example, in hepatocellular carcinoma,, EGR1 can up-regulate SNAIL and SLUG, two important regulators of epithelial-mesenchymal transition (EMT), by activating transcription, thereby promoting the invasion and metastasis ability of liver cancer cells [ 75 ] [ 76 ]. In PC, a 2021 study on pancreatic adenocarcinoma transformation in Science found extensive changes in chromatin openness in repaired pancreatic ductal epithelial cells after pancreatitis remission. Changing the regional peaks can enrich several key transcription factors, including EGR1. In the presence of KRAS mutation, EGR1 wild-type mice have a significantly higher incidence of PC and better survival than EGR1-null mice. shortening, and the activation of EGR1 in this inflammatory-cancer transformation may be mediated by IL-6 [ 77 ]. This shows that EGR1 plays an important role in the development of PC. Combined with our findings, we can continue to explore other functions of EGR1 in PC progression and GEM resistance. Taking a deeper look at the problem of GEM resistance in PC, the failed GEM sensitization clinical trial in PC suggests that a single pathway that induces GEM resistance may not be enough to reverse resistance, and there may be more advanced PC resistance cells. From an epigenetic perspective, changes in chromatin structure and openness are important factors in regulating the transcription of a wide range of genes. The chromatin structure needs to expose the DNA sequence during replication and transcription. This exposed region is the chromatin open region. This region can be combined by transcription factors and other regulatory elements, so the degree of chromatin openness determines the transcription of cellular genes. activity level [ 78 ]. In recent years, following histone modification and DNA modification, chromatin openness has become another key area in epigenetic research. ATAC-seq (Assay for Targeting Accessible-Chromatin with high-throughout sequencing) is used to study staining, the preferred method for qualitative accessibility and openness [ 79 ]. In October 2018, a pan-cancer chromatin openness study based on the ATAC-seq method published in Science showed that the chromatin openness of tumor cells was significantly different from that of normal cells [ 80 ]. Published in "Nature" in February 2021. The research on the mechanism of PC based on single-cell ATAC-seq technology shows that tissue damage and KRAS mutation can induce changes in the degree of chromatin opening of pancreatic cells from an epigenetic perspective, which promotes acinar Ductal metaplasia (ADM) continues to progress to precancerous lesions and even PC [ 81 ]. In recent years, more and more important studies have shown that changes in the degree of chromatin openness are closely related to the treatment sensitivity of various tumors. For example, in BRCA-mutated tumor cells, if the chromatin remodeling protein ALC1 is lost, it will lead to staining In differentiated thyroid cancers with BrafV600E mutations, the loss of the SWI/SNF chromatin remodeling complex induces a decrease in the degree of chromatin openness, resulting in increased sensitivity to MAPK inhibitors. The SY242CS mutation increases chromatin openness in metastatic ER + breast cancer, leading to resistance to aromatase inhibitor therapy in ER + breast cancer patients [ 84 ]. So far, there is no relevant research report on how the degree of chromatin openness changes in the process of GEM drug resistance in PC, and what mechanism is used to induce drug resistance. Therefore, further research on PC from the epigenetic perspective of chromatin openness is required. GEM resistance mechanism and sensitizing GEM efficacy may have better prospects and hope. Conclusions In summary, we have confirmed that PUM2 is a key RNA-binding protein in inducing GEM resistance in PC through in vitro and in vivo experiments and explored the regulatory role of PUM2 on downstream ITGA3 and other focal adhesion-related genes in PC, and its reciprocal regulation relationship with transcription factor EGR1 (Fig. 9 ). It provides clues for the follow-up further study of other biological functions of PUM2 in the occurrence and development of PC, and also provides a new theoretical basis for understanding the GEM resistance of PC and provides a new idea for the treatment effect of sensitizing GEM in PC. Declarations Acknowledgements Not applicable. Author contributions WWB and ZYP designed and supervised the study. ZBB, QC and LZR performed the experiments and collected the data. WYY and LTY contributed to the in vivo experiments. YXY and ZYT contributed to data interpretation. ZBB, QC and LZR performed data analyses. ZBB interpreted the data and wrote the manuscript. All authors read and approved the final manuscript. Funding WWB received support from the National Natural Science Foundation of China (No. 82173074), CAMS Innovation Fund for Medical Sciences (No. 2021-I2M-1-002), National High Level Hospital Clinical Research Funding (No.2022-PUMCH-B-004) and National Multidisciplinary Cooperative Diagnosis and Treatment Capacity Building Project for Major Diseases. ZYP received support from the CAMS Innovation Fund for Medical Sciences (No. 2021-I2M-1-002) and National High Level Hospital Clinical Research Funding (No.2022-PUMCH-D-001). QC received support from the Fundamental Research Fund for the Central Universities (No. 3332022114). The grants supported this study just financially and had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Availability of data and materials The original contributions presented in the study are included in the article/ Supplementary Material. Further inquiries can be directed to the corresponding author. Ethics approval and consent to participate Animal procedures were performed in accordance with the European Community guidelines and were approved by the Institutional Animal Care Committee of “G. d’Annunzio” University and by the Italian Ministry of Health (Authorization n. 892/2018-PR). The study was reviewed and approved by the Ethical Committee of the “G. d’Annunzio” University and Local Health Authority n.2 Lanciano Vasto Chieti, Italy (PROT. 1945/09 COET of 14/07/2009, amended in 2012). The study was performed, after written informed consent from patients, in accordance with the principles outlined in the Declaration of Helsinki. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References SIEGEL R L, MILLER K D, FUCHS H E, et al. Cancer statistics, 2022 [J]. CA Cancer J Clin, 2022, 72(1): 7-33. MIZRAHI J D, SURANA R, VALLE J W, et al. Pancreatic cancer [J]. Lancet, 2020, 395(10242): 2008-20. PUSCEDDU S, GHIDINI M, TORCHIO M, et al. Comparative Effectiveness of Gemcitabine plus Nab-Paclitaxel and FOLFIRINOX in the First-Line Setting of Metastatic Pancreatic Cancer: A Systematic Review and Meta-Analysis [J]. Cancers (Basel), 2019, 11(4). SAIF M W, LEE Y, KIM R. Harnessing gemcitabine metabolism: a step towards personalized medicine for pancreatic cancer [J]. Ther Adv Med Oncol, 2012, 4(6): 341-6. CONROY T, HAMMEL P, HEBBAR M, et al. FOLFIRINOX or Gemcitabine as Adjuvant Therapy for Pancreatic Cancer [J]. N Engl J Med, 2018, 379(25): 2395-406. BINENBAUM Y, NA'ARA S, GIL Z. Gemcitabine resistance in pancreatic ductal adenocarcinoma [J]. Drug Resist Updat, 2015, 23: 55-68. WÖRMANN S M, SONG L, AI J, et al. Loss of P53 Function Activates JAK2-STAT3 Signaling to Promote Pancreatic Tumor Growth, Stroma Modification, and Gemcitabine Resistance in Mice and Is Associated With Patient Survival [J]. Gastroenterology, 2016, 151(1): 180-93.e12. YE Z, HU Q, ZHUO Q, et al. Abrogation of ARF6 promotes RSL3-induced ferroptosis and mitigates gemcitabine resistance in pancreatic cancer cells [J]. Am J Cancer Res, 2020, 10(4): 1182-93. ROSS K C, ANDREWS A J, MARION C D, et al. Identification of the Serine Biosynthesis Pathway as a Critical Component of BRAF Inhibitor Resistance of Melanoma, Pancreatic, and Non-Small Cell Lung Cancer Cells [J]. Mol Cancer Ther, 2017, 16(8): 1596-609. MENG Q, LIANG C, HUA J, et al. A miR-146a-5p/TRAF6/NF-kB p65 axis regulates pancreatic cancer chemoresistance: functional validation and clinical significance [J]. Theranostics, 2020, 10(9): 3967-79. CATENACCI D V, JUNTTILA M R, KARRISON T, et al. Randomized Phase Ib/II Study of Gemcitabine Plus Placebo or Vismodegib, a Hedgehog Pathway Inhibitor, in Patients With Metastatic Pancreatic Cancer [J]. J Clin Oncol, 2015, 33(36): 4284-92. HARTWIG W, STROBEL O, HINZ U, et al. CA19-9 in potentially resectable pancreatic cancer: perspective to adjust surgical and perioperative therapy [J]. Ann Surg Oncol, 2013, 20(7): 2188-96. IOKA T, OKUSAKA T, OHKAWA S, et al. Efficacy and safety of axitinib in combination with gemcitabine in advanced pancreatic cancer: subgroup analyses by region, including Japan, from the global randomized Phase III trial [J]. Jpn J Clin Oncol, 2015, 45(5): 439-48. GLISOVIC T, BACHORIK J L, YONG J, et al. RNA-binding proteins and post-transcriptional gene regulation [J]. FEBS Lett, 2008, 582(14): 1977-86. MOORE K S, VON LINDERN M. RNA Binding Proteins and Regulation of mRNA Translation in Erythropoiesis [J]. Front Physiol, 2018, 9: 910. DICTENBERG J B, SWANGER S A, ANTAR L N, et al. A direct role for FMRP in activity-dependent dendritic mRNA transport links filopodial-spine morphogenesis to fragile X syndrome [J]. Dev Cell, 2008, 14(6): 926-39. LEWIS K, VALANEJAD L, CAST A, et al. RNA Binding Protein CUGBP1 Inhibits Liver Cancer in a Phosphorylation-Dependent Manner [J]. Mol Cell Biol, 2017, 37(16). XU W P, YI M, LI Q Q, et al. Perturbation of MicroRNA-370/Lin-28 homolog A/nuclear factor kappa B regulatory circuit contributes to the development of hepatocellular carcinoma [J]. Hepatology, 2013, 58(6): 1977-91. YE L, LIN S T, MI Y S, et al. Overexpression of LARP1 predicts poor prognosis of colorectal cancer and is expected to be a potential therapeutic target [J]. Tumour Biol, 2016, 37(11): 14585-94. FAGOONEE S, PICCO G, ORSO F, et al. The RNA-binding protein ESRP1 promotes human colorectal cancer progression [J]. Oncotarget, 2017, 8(6): 10007-24. BISH R, VOGEL C. RNA binding protein-mediated post-transcriptional gene regulation in medulloblastoma [J]. Mol Cells, 2014, 37(5): 357-64. BRODY J R, DIXON D A. Complex HuR function in pancreatic cancer cells [J]. Wiley Interdiscip Rev RNA, 2018, 9(3): e1469. COSTANTINO C L, WITKIEWICZ A K, KUWANO Y, et al. The role of HuR in gemcitabine efficacy in pancreatic cancer: HuR Up-regulates the expression of the gemcitabine metabolizing enzyme deoxycytidine kinase [J]. Cancer Res, 2009, 69(11): 4567-72. JIMBO M, BLANCO F F, HUANG Y H, et al. Targeting the mRNA-binding protein HuR impairs malignant characteristics of pancreatic ductal adenocarcinoma cells [J]. Oncotarget, 2015, 6(29): 27312-31. LAL S, CHEUNG E C, ZAREI M, et al. CRISPR Knockout of the HuR Gene Causes a Xenograft Lethal Phenotype [J]. Mol Cancer Res, 2017, 15(6): 696-707. BURKHART R A, PINEDA D M, CHAND S N, et al. HuR is a post-transcriptional regulator of core metabolic enzymes in pancreatic cancer [J]. RNA Biol, 2013, 10(8): 1312-23. ZAREI M, LAL S, PARKER S J, et al. Posttranscriptional Upregulation of IDH1 by HuR Establishes a Powerful Survival Phenotype in Pancreatic Cancer Cells [J]. Cancer Res, 2017, 77(16): 4460-71. BLANCO F F, JIMBO M, WULFKUHLE J, et al. The mRNA-binding protein HuR promotes hypoxia-induced chemoresistance through posttranscriptional regulation of the proto-oncogene PIM1 in pancreatic cancer cells [J]. Oncogene, 2016, 35(19): 2529-41. LI C, JIANG J Y, WANG J M, et al. BAG3 regulates stability of IL-8 mRNA via interplay between HuR and miR-4312 in PDACs [J]. Cell Death Dis, 2018, 9(9): 863. GOLDSTROHM A C, HALL T M T, MCKENNEY K M. Post-transcriptional Regulatory Functions of Mammalian Pumilio Proteins [J]. Trends Genet, 2018, 34(12): 972-90. NÜSSLEIN-VOLHARD C, FROHNHÖFER H G, LEHMANN R. Determination of anteroposterior polarity in Drosophila [J]. Science, 1987, 238(4834): 1675-81. BARKER D D, WANG C, MOORE J, et al. Pumilio is essential for function but not for distribution of the Drosophila abdominal determinant Nanos [J]. Genes Dev, 1992, 6(12a): 2312-26. ZAMORE P D, WILLIAMSON J R, LEHMANN R. The Pumilio protein binds RNA through a conserved domain that defines a new class of RNA-binding proteins [J]. Rna, 1997, 3(12): 1421-33. MURATA Y, WHARTON R P. Binding of pumilio to maternal hunchback mRNA is required for posterior patterning in Drosophila embryos [J]. Cell, 1995, 80(5): 747-56. ZAMORE P D, BARTEL D P, LEHMANN R, et al. The PUMILIO-RNA interaction: a single RNA-binding domain monomer recognizes a bipartite target sequence [J]. Biochemistry, 1999, 38(2): 596-604. SMIALEK M J, ILASLAN E, SAJEK M P, et al. Role of PUM RNA-Binding Proteins in Cancer [J]. Cancers (Basel), 2021, 13(1). NAUDIN C, HATTABI A, MICHELET F, et al. PUMILIO/FOXP1 signaling drives expansion of hematopoietic stem/progenitor and leukemia cells [J]. Blood, 2017, 129(18): 2493-506. JANECKI D M, SAJEK M, SMIALEK M J, et al. SPIN1 is a proto-oncogene and SPIN3 is a tumor suppressor in human seminoma [J]. Oncotarget, 2018, 9(65): 32466-77. GALGANO A, FORRER M, JASKIEWICZ L, et al. Comparative analysis of mRNA targets for human PUF-family proteins suggests extensive interaction with the miRNA regulatory system [J]. PLoS One, 2008, 3(9): e3164. ZHANG C Z, ZHANG J X, ZHANG A L, et al. MiR-221 and miR-222 target PUMA to induce cell survival in glioblastoma [J]. Mol Cancer, 2010, 9: 229. KEDDE M, VAN KOUWENHOVE M, ZWART W, et al. A Pumilio-induced RNA structure switch in p27-3' UTR controls miR-221 and miR-222 accessibility [J]. Nat Cell Biol, 2010, 12(10): 1014-20. VAN ETTEN J, SCHAGAT T L, HRIT J, et al. Human Pumilio proteins recruit multiple deadenylases to efficiently repress messenger RNAs [J]. J Biol Chem, 2012, 287(43): 36370-83. JACKSON R J, HELLEN C U, PESTOVA T V. The mechanism of eukaryotic translation initiation and principles of its regulation [J]. Nat Rev Mol Cell Biol, 2010, 11(2): 113-27. WEIDMANN C A, RAYNARD N A, BLEWETT N H, et al. The RNA binding domain of Pumilio antagonizes poly-adenosine binding protein and accelerates deadenylation [J]. Rna, 2014, 20(8): 1298-319. JOLY W, CHARTIER A, ROJAS-RIOS P, et al. The CCR4 deadenylase acts with Nanos and Pumilio in the fine-tuning of Mei-P26 expression to promote germline stem cell self-renewal [J]. Stem Cell Reports, 2013, 1(5): 411-24. WEIDMANN C A, GOLDSTROHM A C. Drosophila Pumilio protein contains multiple autonomous repression domains that regulate mRNAs independently of Nanos and brain tumor [J]. Mol Cell Biol, 2012, 32(2): 527-40. CHRITTON J J, WICKENS M. A role for the poly(A)-binding protein Pab1p in PUF protein-mediated repression [J]. J Biol Chem, 2011, 286(38): 33268-78. HENTZE M W, MUCKENTHALER M U, ANDREWS N C. Balancing acts: molecular control of mammalian iron metabolism [J]. Cell, 2004, 117(3): 285-97. IVSHINA M, LASKO P, RICHTER J D. Cytoplasmic polyadenylation element binding proteins in development, health, and disease [J]. Annu Rev Cell Dev Biol, 2014, 30: 393-415. HYNES R O. Integrins: a family of cell surface receptors [J]. Cell, 1987, 48(4): 549-54. AOUDJIT F, VUORI K. Integrin signaling in cancer cell survival and chemoresistance [J]. Chemother Res Pract, 2012, 2012: 283181. YANG D, TANG Y, FU H, et al. Integrin β1 promotes gemcitabine resistance in pancreatic cancer through Cdc42 activation of PI3K p110β signaling [J]. Biochem Biophys Res Commun, 2018, 505(1): 215-21. LIU M, ZHANG Y, YANG J, et al. ZIP4 Increases Expression of Transcription Factor ZEB1 to Promote Integrin α3β1 Signaling and Inhibit Expression of the Gemcitabine Transporter ENT1 in Pancreatic Cancer Cells [J]. Gastroenterology, 2020, 158(3): 679-92.e1. SLACK R J, MACDONALD S J F, ROPER J A, et al. Emerging therapeutic opportunities for integrin inhibitors [J]. Nat Rev Drug Discov, 2022, 21(1): 60-78. WANG L, ZHOU W, ZHONG Y, et al. Overexpression of G protein-coupled receptor GPR87 promotes pancreatic cancer aggressiveness and activates NF-κB signaling pathway [J]. Mol Cancer, 2017, 16(1): 61. PRAMANIK K C, FOFARIA N M, GUPTA P, et al. CBP-mediated FOXO-1 acetylation inhibits pancreatic tumor growth by targeting SirT [J]. Mol Cancer Ther, 2014, 13(3): 687-98. ZHOU C, YI C, YI Y, et al. LncRNA PVT1 promotes gemcitabine resistance of pancreatic cancer via activating Wnt/β-catenin and autophagy pathway through modulating the miR-619-5p/Pygo2 and miR-619-5p/ATG14 axes [J]. Mol Cancer, 2020, 19(1): 118. YABUUCHI S, PAI S G, CAMPBELL N R, et al. Notch signaling pathway targeted therapy suppresses tumor progression and metastatic spread in pancreatic cancer [J]. Cancer Lett, 2013, 335(1): 41-51. WOLPIN B M, HEZEL A F, ABRAMS T, et al. Oral mTOR inhibitor everolimus in patients with gemcitabine-refractory metastatic pancreatic cancer [J]. J Clin Oncol, 2009, 27(2): 193-8. QIN C, YANG G, YANG J, et al. Metabolism of pancreatic cancer: paving the way to better anticancer strategies [J]. Mol Cancer, 2020, 19(1): 50. D'AMICO D, MOTTIS A, POTENZA F, et al. The RNA-Binding Protein PUM2 Impairs Mitochondrial Dynamics and Mitophagy During Aging [J]. Mol Cell, 2019, 73(4): 775-87.e10. LEE S, KOPP F, CHANG T C, et al. Noncoding RNA NORAD Regulates Genomic Stability by Sequestering PUMILIO Proteins [J]. Cell, 2016, 164(1-2): 69-80. MUNSCHAUER M, NGUYEN C T, SIROKMAN K, et al. The NORAD lncRNA assembles a topoisomerase complex critical for genome stability [J]. Nature, 2018, 561(7721): 132-6. ELGUINDY M M, KOPP F, GOODARZI M, et al. PUMILIO, but not RBMX, binding is required for regulation of genomic stability by noncoding RNA NORAD [J]. Elife, 2019, 8. KOPP F, ELGUINDY M M, YALVAC M E, et al. PUMILIO hyperactivity drives premature aging of Norad-deficient mice [J]. Elife, 2019, 8. BICKENBACH K A, VEERAPONG J, SHAO M Y, et al. Resveratrol is an effective inducer of CArG-driven TNF-alpha gene therapy [J]. Cancer Gene Ther, 2008, 15(3): 133-9. MARIGNOL L, COFFEY M, HOLLYWOOD D, et al. Radiation to control transgene expression in tumors [J]. Cancer Biol Ther, 2007, 6(7): 1005-12. JEONG S H, KIM H J, RYU H J, et al. ZnO nanoparticles induce TNF-α expression via ROS-ERK-Egr-1 pathway in human keratinocytes [J]. J Dermatol Sci, 2013, 72(3): 263-73. VAISH V, PIPLANI H, RANA C, et al. NSAIDs may regulate EGR-1-mediated induction of reactive oxygen species and non-steroidal anti-inflammatory drug-induced gene (NAG)-1 to initiate intrinsic pathway of apoptosis for the chemoprevention of colorectal cancer [J]. Mol Cell Biochem, 2013, 378(1-2): 47-64. KNUDSEN A M, EILERTSEN I, KIELLAND S, et al. Expression and prognostic value of the transcription factors EGR1 and EGR3 in gliomas [J]. Sci Rep, 2020, 10(1): 9285. FENG Y H, SU Y C, LIN S F, et al. Oct4 upregulates osteopontin via Egr1 and is associated with poor outcome in human lung cancer [J]. BMC Cancer, 2019, 19(1): 791. PARK S Y, KIM J Y, LEE S M, et al. Expression of early growth response gene-1 in precancerous lesions of gastric cancer [J]. Oncol Lett, 2016, 12(4): 2710-5. LIU J, GROGAN L, NAU M M, et al. Physical interaction between p53 and primary response gene Egr-1 [J]. Int J Oncol, 2001, 18(4): 863-70. XIAO D, CHINNAPPAN D, PESTELL R, et al. Bombesin regulates cyclin D1 expression through the early growth response protein Egr-1 in prostate cancer cells [J]. Cancer Res, 2005, 65(21): 9934-42. KUO P L, CHEN Y H, CHEN T C, et al. CXCL5/ENA78 increased cell migration and epithelial-to-mesenchymal transition of hormone-independent prostate cancer by early growth response-1/snail signaling pathway [J]. J Cell Physiol, 2011, 226(5): 1224-31. CHEN H A, KUO T C, TSENG C F, et al. Angiopoietin-like protein 1 antagonizes MET receptor activity to repress sorafenib resistance and cancer stemness in hepatocellular carcinoma [J]. Hepatology, 2016, 64(5): 1637-51. DEL POGGETTO E, HO I L, BALESTRIERI C, et al. Epithelial memory of inflammation limits tissue damage while promoting pancreatic tumorigenesis [J]. Science, 2021, 373(6561): eabj0486. KLEMM S L, SHIPONY Z, GREENLEAF W J. Chromatin accessibility and the regulatory epigenome [J]. Nat Rev Genet, 2019, 20(4): 207-20. BUENROSTRO J D, GIRESI P G, ZABA L C, et al. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position [J]. Nat Methods, 2013, 10(12): 1213-8. CORCES M R, GRANJA J M, SHAMS S, et al. The chromatin accessibility landscape of primary human cancers [J]. Science, 2018, 362(6413). ALONSO-CURBELO D, HO Y J, BURDZIAK C, et al. A gene-environment-induced epigenetic program initiates tumorigenesis [J]. Nature, 2021, 590(7847): 642-8. VERMA P, ZHOU Y, CAO Z, et al. ALC1 links chromatin accessibility to PARP inhibitor response in homologous recombination-deficient cells [J]. Nat Cell Biol, 2021, 23(2): 160-71. SAQCENA M, LEANDRO-GARCIA L J, MAAG J L V, et al. SWI/SNF Complex Mutations Promote Thyroid Tumor Progression and Insensitivity to Redifferentiation Therapies [J]. Cancer Discov, 2021, 11(5): 1158-75. ARRUABARRENA-ARISTORENA A, MAAG J L V, KITTANE S, et al. FOXA1 Mutations Reveal Distinct Chromatin Profiles and Influence Therapeutic Response in Breast Cancer [J]. Cancer Cell, 2020, 38(4): 534-50.e9. Tables Table 1. Correlation between PUM2 expression and clinicopathological parameters in pancreatic cancer Variables PUM2 expression p value † n=44 Low expression （n=16） High expression （n=28） Gender Male 28 10 18 1.000 Female 16 6 10 Age ＜60 23 8 15 1.000 ≥60 21 8 13 Location Head 26 9 17 1.000 Body/Tail 18 7 11 Nerve invasion No 25 9 16 1.000 Yes 19 7 12 Differential dreege G1-2 21 12 11 0.031 G3 23 4 17 T stage T1-2 22 11 9 0.029 T3 22 5 19 N stage N0 25 12 13 0.113 N1-2 19 4 15 TNM stage I/IIA 25 12 13 0.113 IIB/III 19 4 15 Table 2. Univariate and multivariate Cox regression analysis of prognostic risk factors in patients with pancreatic cancer Variabes n Univariate Multivariate HR 95%CI p value HR 95%CI p value Gender Female 15 1.238 0.494-3.102 0.649 Male 31 1 Age ≥60 26 0.562 0.250-1.2611 0.162 ＜60 18 1 Location Body/Tail 18 0.409 0.174-0.964 0.041 2.140 0.281-1.394 0.252 Head 26 1 1 Nerve invasion Yes 16 2.248 0.992-5.097 0.052 No 28 1 Differential degree G3 20 1.984 0.853-4.615 0.112 G1-2 24 1 T stage T3 25 2.253 0.949-5.353 0.066 T1-2 19 1 N stage N1-2 22 2.797 1.263-6.197 0.011 2.140 0.959-4.777 0.063 N0 22 1 1 TNM stage IIB/III 22 2.797 1.263-6.197 0.011 I/IIA 22 1 PUM2 expression High expression 28 16.671 4.882-56.922 ＜0.001 15.280 4.461-52.336 ＜0.001 Low expression 16 1 1 Supplementary Files SupplementaryTable123.docx SupFig1.pdf Supplementary Figure 1. Transcriptome sequencing enrichment of AsPC-1/GEM strain and parent AsPC-1 strain in pancreatic cancer. A, Bubble map of KEGG functional enrichment analysis of differentially expressed AsPC-1/GEM and AsPC-1 genes. B, AsPC-1/GEM significantly higher expression gene KEGG functional enrichment analysis bubble map than AsPC-1. SupFig2.pdf Supplementary Figure 2. Results of MIA PaCa-2 PUM2 KD cells and control cells RNA-seq. A, Correlation analysis between PUM2 KD group samples and control group samples, the results showed that there were 3 samples of PUM2 KD group and control group cells in the intra-group correlation, significantly higher than the inter-group correlation. B, principal component analysis of PUM2 KD group samples and control group samples, the results showed that principal component 1 (PC1) can cluster 3 samples of PUM2 KD group and 3 samples of control group cells respectively. C, volcanic map of differential expression of MIA PaCa-2 PUM2 KD cells and control cells (red: the gene was significantly overexpressed in MIA PaCa-2 PUM2 KD cells compared with control cells; Green: The expression of this gene in MIA PaCa-2 PUM2 KD cells was significantly lower than that in control cells; Blue: there was no significant difference in the expression of this gene in MIA PaCa-2 PUM2 KD cells compared with the control cells). D, MIA PaCa-2 PUM2 KD cells showed significantly higher expression of GO. E, MIA PaCa-2 PUM2 KD cells showed significantly lower expression of GO gene than the control cells. SupFig3.pdf Supplementary Figure 3. KEGG functional enrichment analysis of RNA-seq differentially expressed gene in PUM2 KD cells of MIA PaCa-2 and control cells. A, MIA PaCa-2 PUM2 KD cells and control cell KEGG functional enrichment analysis of all differentially expressed genes bubble map. B, MIA PaCa-2 PUM2 KD cells showed significantly higher expression of KEGG than the control cells. C, MIA PaCa-2 PUM2 KD cells showed significantly lower expression of KEGG gene than the control cells. SupFig4.pdf Supplementary Figure 4. RIP-seq results of PUM2 protein in MIA PaCa-2 cells. A, Distribution of peak on chromosomes of PUM2 antibody IP group by sequencing. B, Peak distribution of PUM2 antibody IP sequencing in the gene functional region. C, distribution of peaks by sequencing of IP group of PUM2 antibody on five transcription functional regions [CDS, 5’utr, 3’utr, transcription start site (TSS) and stop codon]. D, Functional enrichment analysis of intersection genes between peak corresponding genes measured by MIA PaCa-2 PUM2 RIP-seq and genes down-regulated after PUM2 knockdown in RNA-seq. SupFig5.pdf Supplementary Figure 5. The expression of the representative gene of adhesive plaque concentration in pancreatic cancer tissues and its correlation with PUM2 expression. Cite Share Download PDF Status: Published Journal Publication published 17 Feb, 2025 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted Editorial decision: Major Revision 11 Nov, 2024 Reviewers agreed at journal 31 Oct, 2024 Reviewers invited by journal 31 Oct, 2024 Editor assigned by journal 28 Oct, 2024 First submitted to journal 22 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5312328","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":372645133,"identity":"cf984b72-7999-4b62-b0f5-556345410bfb","order_by":0,"name":"Bangbo Zhao","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"prefix":"","firstName":"Bangbo","middleName":"","lastName":"Zhao","suffix":""},{"id":372645134,"identity":"4c176f52-302f-4313-ac8c-097a5febf39c","order_by":1,"name":"Cheng Qin","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Qin","suffix":""},{"id":372645135,"identity":"0281c827-cd01-4598-a2a0-e499f8043114","order_by":2,"name":"Zeru Li","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zeru","middleName":"","lastName":"Li","suffix":""},{"id":372645136,"identity":"c4df621d-7510-4174-aded-8ecba62696fa","order_by":3,"name":"Yuanyang Wang","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuanyang","middleName":"","lastName":"Wang","suffix":""},{"id":372645137,"identity":"39c80865-590f-4d67-b14e-40a929fd4671","order_by":4,"name":"xiaoying Yang","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"prefix":"","firstName":"xiaoying","middleName":"","lastName":"Yang","suffix":""},{"id":372645138,"identity":"42f08817-0ccb-4285-9903-cec1d2ff432b","order_by":5,"name":"Tianyu Li","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tianyu","middleName":"","lastName":"Li","suffix":""},{"id":372645139,"identity":"0f9a8439-9eba-4787-87cb-7aab356c7c9c","order_by":6,"name":"Yutong Zhao","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yutong","middleName":"","lastName":"Zhao","suffix":""},{"id":372645140,"identity":"f199d376-03cd-4b98-a9e2-281b81198573","order_by":7,"name":"Weibin Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApElEQVRIiWNgGAWjYFACHgYGxgYbHn7+BtK0pMlIzjhAmpbDNgYNCURqMLiRe4CZd8d5HgOGA4wfPuYQpSUvgXHmmds85swNzJIztxGhxex2jgHDx7bbPJYNB9iYeYnWkth2jsfgQAIpWj62HSBBi/39d0C/tCXzSM442EycXyR7zgJDrM3Onp+/+eCHj8RoAQL2HxCasYE49aNgFIyCUTAKCAMAr4g16g86zzoAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-6659-9680","institution":"Peking Union Medical College Hospital","correspondingAuthor":true,"prefix":"","firstName":"Weibin","middleName":"","lastName":"Wang","suffix":""},{"id":372645141,"identity":"8efc4acc-4bfe-4a9d-8ae0-97d36860f9c5","order_by":8,"name":"Yupei Zhao","email":"","orcid":"","institution":"Peking Union Medical College Hospital Eastern Branch: Peking Union Medical College Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yupei","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2024-10-22 13:52:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5312328/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5312328/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00018-025-05599-8","type":"published","date":"2025-02-17T15:57:42+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69446600,"identity":"ebea3816-9bcd-498c-8e05-f0a741884d48","added_by":"auto","created_at":"2024-11-20 12:07:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":161859,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePUM2 plays a vital role in PDAC chemoresistance.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA, Cell viability curves of GEM resistant strain AsPC-1/GEM and parent strain AsPC-1 in pancreatic cancer under different GEM concentrations (cell viability = the number of cells in the pore /GEM concentration is the average number of cells in the pore 0, red: AsPC-1/GEM, black: B, transcriptome sequencing volcano map of differential gene between GEM resistant strain AsPC-1/GEM and parent strain AsPC-1 (red: the gene is highly expressed in AsPC-1/GEM; green: the gene is low expressed in AsPC-1/GEM; black: There is no significant difference in the expression of this gene in AsPC-1/GEM and AsPC-1). C, pancreatic cancer is closely related to RBP screening flow chart. D, the ratio of the number of cells in each pore to the control group 72h after transfection of siRNA library in MIA PaCa-2 cells. E, After transfection of PUM2 siRNA cells in the pore and the control cells proliferated for 72h, the central field cells in the pore (DAPI nuclear staining, blue fluorescence treatment to pure black, background treatment to white). F, after transfection of siRNA library in MIA PaCa-2 cells, Cell viability value under gradient concentration GEM (cell viability value = the number of cells in the well /GEM concentration is the average number of cells in the 0 well).g, cell viability value under gradient concentration GEM after transfection of siRNA library in AsPC-1 cells. H, Cell viability curve under gradient concentration GEM in MIA PaCa-2 cells after transfection of siRNA library. I, Cell viability curve under gradient concentration GEM after transfection of siRNA library in AsPC-1 cells. J, the expression level of PUM2 in pancreatic cancer tissues in TCGA database was different from that in normal pancreatic tissues in GTEx database (orange: pancreatic cancer tissues, n=179; Gray: normal pancreatic tissue, n=171). K, disease-free survival curve of patients with high and low PUM2 expression in TCGA database (HR (high PUM2 expression) =1.6, p (HR) =0.003, red, pancreatic cancer patients with high PUM2 expression; Black, pancreatic cancer patients with low PUM2 expression). L, overall survival curve of pancreatic cancer patients with high and low PUM2 expression [red: PUM2 high expression (immunohistochemical score ≥8); black: Low expression of PUM2 (immunohistochemical score \u0026lt; 4)]. M, the expression level of PUM2 protein in GEM resistant strains AsPC-1/GEM was significantly higher than that of parent strains AsPC-1. Student's t-test, *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5312328/v1/0ccc36cfcf8034eedcf79efb.png"},{"id":69448686,"identity":"0f737575-0799-45d2-b745-edb4580fdccb","added_by":"auto","created_at":"2024-11-20 12:23:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":942826,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePUM2 promotes proliferation, migration and chemoresistance of PDAC \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA, the expression levels of PUM2 protein in pancreatic ductal epithelial immortalized cell lines and pancreatic cancer cell lines were detected by Western Blot. B, qRT-PCR was used to detect PUM2 mRNA expression levels in pancreatic duct epithelial immortalized cell lines and pancreatic cancer cell lines. C. Western Blot and qRT-PCR were used to detect the PUM2 knockdown efficiency in MIA PaCa-2 PUM2 stable PUM2 cell line. D, Western Blot and qRT-PCR were used to detect the PUM2 knockdown efficiency in AsPC-1 PUM2 low stable cell lines. E, Western Blot and qRT-PCR were used to detect the overexpression efficiency of PUM2 in BxPC-3 PUM2 stable overexpression cell lines. F, Western Blot and qRT-PCR were used to detect the overexpression efficiency of PUM2 in PANC-1 PUM2 stable overexpression cell lines. G, PUM2 knockdown, the proliferation ability of MIA PaCa-2 cells was significantly decreased. H, PUM2 was knocked down, and the proliferation ability of AsPC-1 cells was significantly decreased. I, overexpression of PUM2, BxPC-3 cell proliferation significantly increased. J, overexpression of PUM2, PANC-1 cell proliferation significantly increased. K, PUM2 knockdown, MIA PaCa-2 cells were significantly more sensitive to GEM. L, PUM2 was knocked down, and the sensitivity of AsPC-1 cells to GEM was significantly increased. M, overexpression of PUM2, BxPC-3 cells were significantly less sensitive to GEM. N, overexpression of PUM2, PANC-1 cells were significantly less sensitive to GEM. O, PUM2 knockdown, MIA PaCa-2 cell migration ability decreased significantly. P, PUM2 knockdown, AsPC-1 cell migration ability decreased significantly. Q, PUM2 overexpression, BxPC-3 cell migration ability increased significantly. R, overexpression of PUM2, PANC-1 cell migration ability increased significantly. Student's t - test, and * * * * * p \u0026lt; 0.01, p \u0026lt; 0.005, p \u0026lt; 0.001. * * * *.\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5312328/v1/c96180d47db32fc22a2ee80a.png"},{"id":69447839,"identity":"0c7fd77c-9e4e-412b-897b-b047170b275f","added_by":"auto","created_at":"2024-11-20 12:15:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1132384,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePUM2 promotes proliferation, metastasis and chemoresistance of PDAC \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA, general picture of nude mice after 6 weeks of tumor formation in NC+PBS, shPUM2+PBS, NC+GEM and shPUM2+GEM groups. B, tumor volume growth curve of subcutaneous grafts in nude mice in NC+PBS, shPUM2+PBS, NC+GEM and shPUM2+GEM groups. C, NC+PBS, shPUM2+PBS, NC+GEM, and shPUM2+GEM groups were sacrificed 6 weeks after tumor formation, and the gross figure of the transplanted tumor was dissected. D, Statistical results of transplanted tumor weight 6 weeks after tumor formation in NC+PBS, shPUM2+PBS, NC+GEM and shPUM2+GEM groups. Student's t-test, *p\u0026lt;0.05; ****p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5312328/v1/1b37a770c15a133c96fbba1b.png"},{"id":69447846,"identity":"956d44b7-f36d-48c7-a1ae-e9be81a2d836","added_by":"auto","created_at":"2024-11-20 12:15:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":313131,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNA-seq and RIP-seq jointly reveals RNA regulating function of PUM2 in PDAC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA, Heat maps of differentially expressed genes in MIA PaCa-2 PUM2 KD cells and control cells (red: highly expressed; Green: low expression). B, MIA PaCa-2 PUM2 KD cells were significantly lower than the control cells KEGG functional enrichment analysis bubble map. C, GSEA analysis of gene expression in MIA PaCa-2 PUM2 KD cells and adhesive patch gene set. D. KEGG function prediction analysis of the peak gene corresponding to PUM2 antibody IP group sequencing. E, in MIA PaCa-2 cells, PUM2 antibody IP group showed the difference of peak corresponding gene and MIA PaCa-2 PUM2 KD cells showed significantly lower gene expression than the control group. In F, PUM2 IP, adhesive patch genes concentrated represented the abundance of mRNA fragments bound by PUM2 antibody.\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5312328/v1/a3baad64a090215663516be8.png"},{"id":69447840,"identity":"dda2ea9e-55e1-466a-8663-9e1bef442e8d","added_by":"auto","created_at":"2024-11-20 12:15:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":152471,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFocal adhesion pathway is regulated integrally by PUM2 at mRNA level in PDAC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA, PUM2 was knocked down in MIA PaCa-2 cells, and the mRNA level of adhesion plaque related genes was significantly decreased. B, PUM2 was knocked down in AsPC-1 cells, and the mRNA level of adhesion plaque related genes was significantly decreased. PUM2 was highly expressed in C, BxPC-3 cells, and the mRNA level of adhesion plaque related genes was significantly increased. D, PUM2 was highly expressed in PANC-1 cells, and the mRNA level of sticking-plaque related genes was significantly increased. E, RIP-qPCR indicated that the 3’utr region fragment of the plaque associated gene was significantly enriched in the RNA bound by PUM2 protein in MIA PaCa-2 cells. F, agarose gel electrophoresis image obtained from qRT-PCR product in Figure (E). G, RIP-qPCR indicated that the 3’UTR region fragment of the plaque associated gene was significantly enriched in the Pum2-bound RNA in AsPC-1 cells. H, agarose gel electrophoresis image obtained from qRT-PCR products in (G). In I, MIA PaCa-2 cells, the mRNA stability of ITGA3, ADAM17, FLNA and COL1A2 associated with adhesion plaques decreased significantly after PUM2 knockdown. J, in AsPC-1 cells, the mRNA stability of ITGA3, ADAM17, ASAP1, FLNA, COL1A2 was significantly decreased after PUM2 knockdown. Student's t-test; * p \u0026lt; 0.05; **p \u0026lt; 0.01; * * * p \u0026lt; 0.005; * * * * p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5312328/v1/7abc72ceb2f8b8d4e4ad5d0b.png"},{"id":69446605,"identity":"ce4330b3-6c9b-462a-8da4-56c8e86c4e99","added_by":"auto","created_at":"2024-11-20 12:07:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":635383,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eITGA3 is a crucial downstream in PUM2 promoting chemoresistance and metastasis of PDAC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA, the 3’utr of ITGA3 gene contains the PUM binding element (PBE) sequence. In B, MIA PaCa-2 cells, after PUM2 knockdown, the expression degree of downstream genes in ITGA3 3’utr PBE sequence wild type plasmid was significantly decreased, while the expression degree of downstream genes in ITGA3 3’utr PBE sequence mutant plasmid was not significantly changed. In C, MIA PaCa-2 and AsPC-1 cells, the expression level of ITGA3 and pAKT decreased significantly after PUM2 knockdown, while the level of total AKT did not change significantly. In D, BxPC-3 and PANC-1 cells, the expression level of ITGA3 and pAKT was significantly higher after PUM2 overexpression, and the level of total AKT was significantly increased. E, the protein expression levels of PUM2 and ITGA3 in NC, shPUM2, shPUM2+Vec, shPUM2+ITGA3 groups were verified by Western Blot. In F, MIA PaCa-2 cells, shPUM2 group was significantly more sensitive to GEM than NC group. After overexpression of ITGA3, shPUM2+ITGA3 group was significantly more sensitive to GEM than shPUM2 group. In G, AsPC-1 cells, the GEM sensitivity of shPUM2 group was significantly increased compared with NC group. After overexpression of ITGA3, the GEM sensitivity of shPUM2+ITGA3 group was significantly restored compared with shPUM2 group. H, in MIA PaCa-2 cells, the cell migration ability of shPUM2 group was significantly decreased compared with NC group. After overexpression of ITGA3, the cell migration ability of shPUM2+ITGA3 group was significantly recovered compared with shPUM2 group, and there was no significant difference between the SHPUM2 and NC group. I, in AsPC-1 cells, the cell migration ability of shPUM2 group was significantly decreased compared with NC group. After overexpression of ITGA3, the cell migration ability of shPUM2+ITGA3 group was significantly recovered compared with shPUM2 group. Student's t-test, ns p \u0026gt; 0.05; * * p \u0026lt; 0.01; * * * * p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5312328/v1/af73c7b0d50adf662ea24a28.png"},{"id":69446610,"identity":"d6919fff-2169-47b1-a458-704dcf8bcbab","added_by":"auto","created_at":"2024-11-20 12:07:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":297352,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEGR1 is a potential downstream transcription factor of PUM2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA, the possible upstream transcription factors of PUM2 predicted by JASPAR database (366) and the mRNA corresponding genes of PUM2 binding in RIP-seq (1535) were intersected with the genes whose expression was down-regulated after PUM2 knockdown in RNA-seq (1966). B, the expression level of EGR1 in pancreatic cancer tissues (n=179) was significantly higher than that in normal pancreas tissues (n=171). C, the expression level of EGR1 in pancreatic cancer tissues was correlated with the expression level of PUM2. D, RBP prediction of possible binding of EGR1 mRNA based on Postar.com. Student's t-test, *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-5312328/v1/e24a6fe10b5f8370ad45e4d6.png"},{"id":69447844,"identity":"573f3425-1d3b-4713-86af-e74e3187c30c","added_by":"auto","created_at":"2024-11-20 12:15:48","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":450809,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEGR1 and PUM2 regulate mutually in PDAC resulting in a cascaded effect.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA, qRT-PCR results showed that the mRNA level of EGR1 was significantly decreased in MIA PaCa-2 and AsPC-1 cells when PUM2 was knocked down. B, Western Blot results showed that PUM2 knockdown in MIA PaCa-2 and AsPC-1 cells significantly reduced EGR1 protein levels. C, qRT-PCR results showed that PUM2 was overexpressed in BxPC-3 and PANC-1 cells, and the mRNA level of EGR1 was significantly increased. D, Western Blot results showed that PUM2 was overexpressed in BxPC-3 and PANC-1 cells, and the level of EGR1 protein was significantly increased. E, qRT-PCR results showed that PUM2 mRNA levels significantly decreased after EGR1 knockdown in BxPC-3 cells. F, Western Blot results showed that EGR1 knockdown, PUM2 and ITGA3 protein levels were significantly decreased in BxPC-3 cells. G, qRT-PCR results showed that the overexpression of EGR1 in PANC-1 cells significantly increased the mRNA level of PUM2. H, Western Blot results showed that EGR1 was overexpressed in PANC-1 cells, and the protein levels of PUM2 and ITGA3 were significantly increased. I, RIP-qPCR indicated that fragments in the 3’utr region of EGR1 mRNA were significantly enriched in the RNA bound to PUM2 protein in MIA PaCa-2 cells. Below is the agarose gel electrophoresis image of the qRT-PCR product in the upper bar chart. J, RIP-qPCR indicated that fragments in the 3’utr region of EGR1 mRNA were significantly enriched in the Pum2-bound RNA in AsPC-1 cells. Below is the agarose gel electrophoresis image of the qRT-PCR product in the upper bar chart. K, Western Blot was used to verify the protein expression levels of PUM2 and ITGA3 in NC, shPUM2, shPUM2+Vec, shPUM2+EGR1 groups. L, MIA PaCa-2 cells, shPUM2 group was significantly more sensitive to GEM than NC group, after overexpression of ITGA3, shPUM2+ITGA3 group was significantly more sensitive to GEM than shPUM2 group. In M and MIA PaCa-2 cells, the migration ability of shPUM2 group was significantly decreased compared with NC group. After overexpression of ITGA3, the migration ability of shPUM2+ITGA3 group was significantly recovered compared with shPUM2 group. Student's t-test, *p \u0026lt; 0.05; **p \u0026lt; 0.01; * * * p \u0026lt; 0.005; * * * * p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-5312328/v1/6ceabcc24495e9b30b7a3466.png"},{"id":69446603,"identity":"208748a5-91b7-408e-a071-54e552645695","added_by":"auto","created_at":"2024-11-20 12:07:48","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":701223,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of EGR1-PUM2-ITGA3 axis in PDAC chemoresistance and invasiveness. \u003c/strong\u003ePUM2 protein is highly expressed in pancreatic cancer, and can bind to the 3’utr region of ITGA3 and other sticking-plaque related genes mRNA, improve the stability of the above mRNA and up-regulate the expression of the above gene, so as to play the function of promoting GEM resistance and promoting cell migration. PUM2 can also bind to the 3’utr region of transcription factor EGR1 mRNA, and EGR1 can also act as a transcription factor to regulate the transcription level of PUM2, forming an interregulatory relationship, thereby amplifies the function of PUM2 in promoting GEM resistance and cell migration in pancreatic cancer.\u003c/p\u003e","description":"","filename":"fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-5312328/v1/fed2fdff0007f92bcdcca76e.png"},{"id":77054737,"identity":"50afd223-46bb-49a0-a90c-92b4a40b0390","added_by":"auto","created_at":"2025-02-24 16:31:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6428291,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5312328/v1/0e2268fe-2352-4ca4-9b91-2ebc62afb712.pdf"},{"id":69446597,"identity":"99466f9e-2a73-4cc7-942c-d76c399eacdd","added_by":"auto","created_at":"2024-11-20 12:07:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":25909,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable123.docx","url":"https://assets-eu.researchsquare.com/files/rs-5312328/v1/b5d415cbc694159b8d2adc07.docx"},{"id":69446598,"identity":"7b2e2c46-2e50-4c4b-b91c-6f0de23fdf84","added_by":"auto","created_at":"2024-11-20 12:07:48","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":49023,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. Transcriptome sequencing enrichment of AsPC-1/GEM strain and parent AsPC-1 strain in pancreatic cancer.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA, Bubble map of KEGG functional enrichment analysis of differentially expressed AsPC-1/GEM and AsPC-1 genes. B, AsPC-1/GEM significantly higher expression gene KEGG functional enrichment analysis bubble map than AsPC-1.\u003c/p\u003e","description":"","filename":"SupFig1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5312328/v1/c4ed4441926dd52567c6d580.pdf"},{"id":69446602,"identity":"fefdaeed-2920-4dc2-8e0c-f7f0208fc15e","added_by":"auto","created_at":"2024-11-20 12:07:48","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":390461,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2. Results of MIA PaCa-2 PUM2 KD cells and control cells RNA-seq.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA, Correlation analysis between PUM2 KD group samples and control group samples, the results showed that there were 3 samples of PUM2 KD group and control group cells in the intra-group correlation, significantly higher than the inter-group correlation. B, principal component analysis of PUM2 KD group samples and control group samples, the results showed that principal component 1 (PC1) can cluster 3 samples of PUM2 KD group and 3 samples of control group cells respectively. C, volcanic map of differential expression of MIA PaCa-2 PUM2 KD cells and control cells (red: the gene was significantly overexpressed in MIA PaCa-2 PUM2 KD cells compared with control cells; Green: The expression of this gene in MIA PaCa-2 PUM2 KD cells was significantly lower than that in control cells; Blue: there was no significant difference in the expression of this gene in MIA PaCa-2 PUM2 KD cells compared with the control cells). D, MIA PaCa-2 PUM2 KD cells showed significantly higher expression of GO. E, MIA PaCa-2 PUM2 KD cells showed significantly lower expression of GO gene than the control cells.\u003c/p\u003e","description":"","filename":"SupFig2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5312328/v1/2f506aeb1f3702ffbc5934e3.pdf"},{"id":69448706,"identity":"06e00caa-80b4-4adf-9c0c-d6afbde437fd","added_by":"auto","created_at":"2024-11-20 12:23:48","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":70422,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3. KEGG functional enrichment analysis of RNA-seq differentially expressed gene in PUM2 KD cells of MIA PaCa-2 and control cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA, MIA PaCa-2 PUM2 KD cells and control cell KEGG functional enrichment analysis of all differentially expressed genes bubble map. B, MIA PaCa-2 PUM2 KD cells showed significantly higher expression of KEGG than the control cells. C, MIA PaCa-2 PUM2 KD cells showed significantly lower expression of KEGG gene than the control cells.\u003c/p\u003e","description":"","filename":"SupFig3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5312328/v1/2ece09bed875644b78488374.pdf"},{"id":69446609,"identity":"62433c33-470a-40d9-9acf-52ce0d8776f3","added_by":"auto","created_at":"2024-11-20 12:07:48","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":770981,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 4. RIP-seq results of PUM2 protein in MIA PaCa-2 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA, Distribution of peak on chromosomes of PUM2 antibody IP group by sequencing. B, Peak distribution of PUM2 antibody IP sequencing in the gene functional region. C, distribution of peaks by sequencing of IP group of PUM2 antibody on five transcription functional regions [CDS, 5’utr, 3’utr, transcription start site (TSS) and stop codon]. D, Functional enrichment analysis of intersection genes between peak corresponding genes measured by MIA PaCa-2 PUM2 RIP-seq and genes down-regulated after PUM2 knockdown in RNA-seq.\u003c/p\u003e","description":"","filename":"SupFig4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5312328/v1/a95ddd4e0ba3db6c284f25e9.pdf"},{"id":69447841,"identity":"dd23ae89-a4f0-4f1e-a1e0-cf7b27daadf3","added_by":"auto","created_at":"2024-11-20 12:15:48","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":198230,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 5. The expression of the representative gene of adhesive plaque concentration in pancreatic cancer tissues and its correlation with PUM2 expression.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"SupFig5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5312328/v1/7470ac882b26d98b606ef970.pdf"}],"financialInterests":"","formattedTitle":"RNA binding protein Pumilio2 promotes chemoresistance of pancreatic cancer via focal adhesion pathway and interacting with transcription factor EGR1","fulltext":[{"header":"Background","content":"\u003cp\u003ePancreatic ductal adenocarcinoma (hereinafter referred to as pancreatic cancer, PC) has insidious onset, rapid progression and extremely poor prognosis. Up to now, the 5-year survival rate is still less than 10% [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Surgical resection is the only possible way to cure pancreatic cancer. However, due to the lack of clear clinical symptoms or signs in the early stage, 80\u0026ndash;85% of pancreatic cancer patients have lost the chance of surgical resection when diagnosed [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. For these patients with advanced stage or distant metastasis, chemotherapy is the most important treatment plan. The latest NCCN Clinical Practice Guideline for pancreatic cancer (version 2022.1) recommends that patients with pancreatic cancer receive chemotherapy regardless of pathological stage. Gemcitabine (GEM) based combination regimen is the most widely used first-line treatment regimen in clinical chemotherapy of pancreatic cancer at present [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. GEM is a nucleoside antitumor drug that antagonizes nucleotide metabolism, and its derivatives can interfere with DNA synthesis and cell cycle to induce apoptosis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, GEM resistance in patients with pancreatic cancer is the biggest practical difficulty in clinical work at present. Taking postoperative adjuvant chemotherapy as an example, the proportion of GEM resistance within 3 years is as high as 78.6%. Even so, due to the poor efficacy or excessive toxicity of many other regimens, Gem-based chemotherapy is still the preferred chemotherapy regimen for most patients with pancreatic cancer [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, it is of great clinical significance to explore the mechanism of GEM resistance in pancreatic cancer to enhance the sensitization effect and reverse the drug resistance.\u003c/p\u003e \u003cp\u003eThe mechanism of GEM drug resistance is very complex, which is closely related to tumor microenvironment, instability of genetic material, regulation of intracellular signaling molecules, etc. Activation or inactivation of classical intracellular pathways is one of the main mechanisms causing primary and acquired drug resistance [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. For example, inactivated p53 induces GEM drug resistance by activating the proliferation-promoting JAK2-STAT3 signaling pathway [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The downstream ARF6 protein of Kras/ERK signaling pathway can enhance the drug resistance of GEM by down-regulating the expression of dCK and hENT1 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Gem-resistant pancreatic cancer cells can up-regulate the expression of tryptophan synthetase through MAPK signaling pathway and promote the synthesis of tryptophan to achieve metabolic reprogramming [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Mir-146a-5p can mediate GEM resistance of pancreatic cancer by activating NF-κB pathway [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. At present, some therapeutic strategies have been used in combination with GEM to block the signaling pathway and reverse or delay the development of drug resistance, but most of them are ineffective. For example, Vismodegib plays an inhibitory role in cancer by inhibiting Hedgehog signaling pathway. In a phase II clinical trial, the combination regimen of Vismodegib and GEM did not significantly improve progression-free survival and overall survival of patients with metastatic pancreatic cancer compared with GEM alone (NCT01064622) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Ibrutinib is a tyrosine kinase inhibitor. Although it has shown good safety in phase I/II clinical trials [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], in phase III clinical trials, Ibrutinib combined with chemotherapy still failed to show significant advantages in improving progression-free survival or overall survival (NCT02436668). In phase III clinical trials of Axitinib, a selective inhibitor of vascular endothelial growth factor receptor, GEM combined with Axitinib did not provide additional survival benefit for patients with advanced pancreatic cancer compared with monotherapy (NCT00471146) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These failed clinical trials suggest that our understanding of the mechanism of GEM in pancreatic cancer is still limited, and the role of post-transcriptional regulation, post-translational modification and epigenetics in GEM resistance is still unknown. Therefore, starting from the above emerging gene molecular biological regulatory mechanisms, further study of GEM resistance mechanism and reversal of GEM resistance has a good prospect and hope.\u003c/p\u003e \u003cp\u003eRNA Binding Protein (RBP) is a Protein that can bind to single or double stranded RNA molecules. RBPS usually contain at least one RNA binding domain (RBD) and are divided into different families according to the different RBDS contained. Currently known RBDS include: RNA recognition module (RBM), KH domain, double-stranded RNA binding domain (dsRBD), zinc finger protein domain (ZnF), PAZ domain, PIWI domain, SAM domain, etc. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. There are a large number of RBPS, accounting for about 6\u0026ndash;8% of the protein coding genes in cells. At present, a total of 1171 eukaryotic RBPS have been recorded in RBP Database. RBPS bind to various Rnas and are widely involved in various stages of RNA molecular metabolism, which determines the fate of RNA from synthesis to decomposition. It plays an important biological role in cell development and cell metabolism. For example, mRNA splicing is the first step of RNA post-transcriptional processing, and the spliceosome that catalyzes this process is the RNA-protein complex formed by RBP. PolyA (polyadenylate) tail is an important component of eukaryotic mRNA, which plays an important role in mRNA nucleation, shearing, stability and translation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The Cleavage and Polyadenylation Specificity Factor CPSF (RNA binding protein) is required in almost all mRNA tail adding processes. CPSF can bind to AAUAAA sequence and recruit and activate ployA polymerase activity together with ployA binding protein [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Another example is that mRNA is transferred from nucleus to cytoplasm after processing and maturation. TAP/NXF1 heterodimer is a key molecule mediating this process, but TAP cannot interact directly with RNA, so Aly/REF, an RNA-binding protein, is also required to participate. In addition, some RBPS (ZBP1, FMRP, etc.) can also mediate the intracellular localization of mRNA to achieve the specificity of the spatial localization of corresponding proteins [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In addition, mRNA translation in ribosomes is the last step of mRNA to produce functional proteins, and RBP is not only an important component of ribosomes, but also many RBPS regulate the translation of specific mrnas by recruiting or rejecting translation initiation complexes. Therefore, RBP is an important partner of mRNA, regulating all the key processes of mRNA molecule cleavage, editing, transport, degradation and translation, and is necessary for mRNA maturation and proper function. At the same time, RBP is also important for the generation and biological function of non-coding RNA. Therefore, RBP plays an indispensable role in a variety of life activities, and its abnormal expression or mutation will lead to the occurrence of a variety of diseases.\u003c/p\u003e \u003cp\u003eAt present, the biological function of RBP in the occurrence and development of pancreatic cancer is gradually being revealed, and many important findings and achievements have been made in the research of liver cancer, colorectal cancer and nervous system tumors [\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Dozens of RBPS such as RNA-binding proteins HuR, CUGBP2, CPEB and IMP3 have been shown to regulate the proliferation, invasion and chemotherapy resistance of pancreatic cancer cells [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Taking HuR as an example, Costantino Tantamount first reported the high expression of HuR in pancreatic cancer cells in 2009, and the sensitivity of pancreatic cancer cells with high expression of HuR to GEM was significantly enhanced [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Jimbo et al. found that knockdown of HuR expression by shRNA could significantly reduce the invasion and metastasis ability of pancreatic cancer cells, whereas overexpression of HuR could enhance the invasion and metastasis ability of pancreatic cancer cells [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Lal et al. obtained HUR-null pancreatic cancer cell line by double knockout of HuR gene and found that the proliferation ability of HuR (-/-) cells was significantly decreased, and the tumorigenicity of HuR (-/-) pancreatic cancer cells in mice was also significantly decreased [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Other studies have shown that pancreatic cancer cells can survive and proliferate in the pancreatic cancer microenvironment of chronic hypoxia and nutrient deprivation by activating hypoxia inducible factors (HIFs) such as HuR [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Zarei et al. recently found that the regulatory axis of HuR-IDH1 is a key regulatory protein in the microenvironment of pancreatic cancer in the hypotrophic state, which can be used as a potential therapeutic target [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In addition, Blanco et al. demonstrated that HuR could promote the chemoresistance of pancreatic cancer cells in anaerobic environment by regulating the stability of PIM1mRNA, and the application of HuR inhibitor ms-444 could reverse the chemoresistance of tumor cells [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Li et al. demonstrated that HuR promoted the stability of IL-8 mRNA by regulating Mir-4312 translation and down-regulated the expression of BAG3 in pancreatic cancer cells, thus significantly reducing the migration and invasion ability of tumor cells [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, even with these findings, we still know little about the function, molecular mechanism and expression regulation of many other RBPS in pancreatic cancer, especially GEM resistance in pancreatic cancer. Therefore, it is of great theoretical significance and clinical value to systematically identify the key RBPS involved in GEM resistance of pancreatic cancer, reveal the specific molecular mechanism involved in the occurrence of drug resistance, and develop new chemotherapy drug screening strategies and sensitization strategies for pancreatic cancer based on this target.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003ePDAC clinical specimens and cell lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePancreatic ductal adenocarcinoma tissue was obtained from 82 PC patients who underwent operation in Peking Union Medical College Hospital and the pathological diagnoses were made by two pathologists independently. Clinical and pathological data were extracted from the medical record system of our center. Informed consents were obtained from each patient and the collection were approved by the Ethics Committee of Peking Union Medical College Hospital. PC cell lines used in our study were purchased from American Type Culture Collection (ATCC) except for AsPC-1/GEM, which was constructed by our research group previously. HPNE, MIA PaCa-2, PANC-1 and T3M4 were cultured in Dulbecco\u0026apos;s Modified Eagle Medium (DMEM)/High Glucose (Hyclone). AsPC-1, ASPC-1/GEM and SW1990 were cultured in RPMI-1640 modified medium (Hyclone). BxPC-3 was cultured in RPMI (Corning). Capan-1 and CFPAC were cultured in Iscove\u0026apos;s Modified Dubecco\u0026apos;s Medium (IMDM, Hyclone). The culture medium of Capan-1 was added with 20% fetal bovine serum (FBS) and the culture medium of the other cell lines were added with 10% FBS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003esiRNA library screening\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eScreening associated siRNA were designed and synthesized by RIBOBIO (Guangzhou, China). Three different siRNAs were designed targeting different sequences of a single gene, which were then mixed up before transfection. Reverse transfection were conducted in 96-well board using Lipofectamine\u003csup\u003eTM\u003c/sup\u003e 3000 (ThermoFisher, US). The transfection system was composed of 0.2\u0026mu;g siRNA mix, 0.2\u0026mu;L LipofectamineTM 3000 and 5000 cells. GEM was added after 24h of the seeding and the 96-well board was then cultured for another 48h before counting.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemical analyses were conducted according to standard procedures. Briefly, after deparaffinization, rehydration, antigen retrieval and endogenous peroxidase blockage, sections were incubated with anti-PUM2 antibody (1:100 dilution, Abcam, USA) at 4 \u0026deg;C overnight. Subsequently, sections were washed by PBS and incubated with HRP‐labeled secondary antibody for 30 min. After application of diaminobenzidine as a chromogen, the slides were evaluated using light microscopy (Olympus, Japan). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePUM2 stable overexpression and knockdown cell lines construction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the function of PUM2, full-length human PUM2 cDNA was cloned into the pLentiGV492 expression vector (Genechem, Shanghai). For PUM2 knockdown assay, two short hairpin RNAs (shRNA) specifically targeting RALYL were cloned into the GV 493 lentiviral vector (Genechem, Shanghai). Stably transduced cells were selected by puromycin (Sigma-Aldrich). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro functional assays and animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded into 96-well plates for 8 hours before adding chemotherapeutic drugs. The concentration gradients of drugs were 0, 1nM, 10nM, 100nM, 1\u0026mu;M, 10\u0026mu;M, 100\u0026mu;M and 1M. After treating for 48 hours, drug-containing culture medium was replaced by fresh medium which contained 10% CCK-8 (Dojindo, Japan). Place the plates in incubator under 37℃ for 2 hours and determine its light absorption value at 450nm and 630nm using an enzyme-linked immunosorbent detector (Invitrogen, Thermo Fisher Scientific, USA). The difference between absorbance values at 450nm and 630nm indirectly reflects the number of living cells.Cell viability values under the drug concentration gradients were conducted non-linear fitting (inhibitor, four parameters) and IC50 was defined as the drug concentration where slope of the fit curve came to maximum (GraphPad Prism 9.0). A nude mouse xenograft model was used to evaluate tumor formation and chemoresistant ability in vivo. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction and quantitative real-time PCR (qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNAs were extracted from cultured cell lines by Trizol reagent (Invitrogen, USA). Then cDNA was synthesized using the TaqMan Reverse Transcription Kit (Takara, Dalian, China) according to the manufacturer\u0026apos;s instructions. For mRNA analysis, qRT-PCR was performed with SYBR\u0026reg; Premix Ex TaqTM Reagent (TaKaRa, Dalian, China) by using StepOne Plus Real-Time PCR system (Applied Biosystems, USA). Fold changes relative to \u0026beta;-actin were calculated using 2\u0026minus;\u0026Delta;\u0026Delta;Ct method. All primer sequences were listed in Supplementary Table 3.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantified protein lysates were resolved on SDS-PAGE, transferred onto a polyvinylidenedifluoride (PVDF) membrane (Millipore), and then blocked with 5% non-fat milk in Tris-buffered saline-Tween 20 (TBS-T) for 1 h at room temperature. The blocked membrane was then incubated with primary antibody diluted 1:1000 in 5% bovine serum albumin in TBS-T at 4 \u0026deg;C overnight. All antibodies used are listed in Supplementary Table 4. After washing with TBS-T, the membrane was incubated for 1 h with horseradish peroxidase HRP-conjugated secondary antibody. A complex of primary and secondary antibodies- labeled proteins were detected by enhanced chemiluminescence (ECL) system followed by exposure to 5200Multi visualizer (Tanon, China). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Total RNA was used as input material for the RNA sample preparations.\u003c/p\u003e\n\u003cp\u003eBriefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase, then use RNaseH to degrade the RNA. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and dNTP. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3\u0026rsquo; ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing (Novogene Experimental Department)\u003c/p\u003e\n\u003cp\u003eThe clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer\u0026rsquo;s instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated.\u003c/p\u003e\n\u003cp\u003eDifferential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq2 R package (1.20.0). DESeq2 provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg\u0026rsquo;s approach for controlling the false discovery rate. Genes with an adjusted P-value \u0026lt;0.05 found by DESeq2 were assigned as differentially expressed. Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the clusterProfiler R package, in which gene length bias wascorrected. GO terms with corrected Pvalue less than 0.05 were considered significantly enriched by differential expressed genes. KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-through put experimental technologies (http://www.genome.jp/kegg/). We used clusterProfiler R package to test the statistical enrichment of differential expression genes in KEGG pathways. The Reactome database brings together the various reactions and biological pathways of human model species. Reactome pathways with corrected Pvalue less than 0.05 were considered significantly enriched by differential expressed genes. The DO (Disease Ontology) database describes the function of human genes and diseases. DO pathways with corrected Pvalue less than 0.05 were considered significantly enriched by differential expressed genes. The DisGeNET database integrates human disease-related genes. DisGeNET pathways with corrected Pvalue less than 0.05 were considered significantly enriched by differential expressed genes. We used clusterProfiler software to test the statistical enrichment of differentially expressed genes in the Reactome pathway, the DO pathway, and the DisGeNET pathway. Gene Set Enrichment Analysis (GSEA) is a computational approach to determine if a pre- defined Gene Set can show a significant consistent difference between two biological states. The genes were ranked according to the degree of differential expression in the two samples, and then the predefined Gene Set were tested to see if they were enriched at the top or bottom of the list. Gene set enrichment analysis can include subtle expression changes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA immunoprecipitation (RIP)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA immunoprecipitation (RIP) experiment was performed using Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) according to the manufacturer\u0026apos;s instructions. Briefly, PC cells were lysed by RIP lysis buffer and then cell lysates were immunoprecipitated with protein A/G magnetic beads conjugated to anti-PUM2 anti-body (Abcam, USA) or normal rabbit IgG at 4 \u0026deg;C overnight. After RNA purification, qRT-PCR was used to measure the levels of focal adhesion pathway genes transcripts in the protein-RNA complexes. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRIP sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLibrary Preparation and Quantification\u003c/p\u003e\n\u003cp\u003eThe library was constructed by Novogene Corporation (Beijing, China). Subsequently, pair-end sequencing of sample was performed on Illumina platform (Illumina, CA, USA). Library quality was assessed on the Agilent Bioanalyzer 2100 system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw data (raw reads) of fastq format were firstly processed using fastp (version 0.19.11, Chen et al., 2018) software. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low-quality reads from raw data. At the same time, Q20, Q30 and GC content of the clean data were calculated. All the downstream analyses were based on the clean data with high quality. Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using BWA (v 0.7.12) and clean reads were aligned to the reference genome using BWA mem (v 0.7.12). After mapping reads to the reference genome, we used the MACS2(version 2.1.0) peak calling software to identify regions of IP enrichment over background. A q-value threshold of 0.05 was used for all data sets. After peak calling, the distribution of chromosome distribution, peak width, fold enrichment, significant level and peak summit number per peak were all displayed. The interaction between transcript factor or chromatin histone modification and DNA were not random, while they show some specific sequence preference. Homer (Heinz et al., 2010) was used to detect the denovo sequence motif and the matched known motifs. Peak related genes can be confirmed by PeakAnnotator, and then Gene Ontology (GO) enrichment analysis of performed to identify the function enrichment results. GO enrichment analysis was implemented by the GOseq R package, in which gene length bias wascorrected. GO terms with corrected Pvalue less than 0.05 were considered significantly enriched by peak related genes. KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-through put experimental technologies (http://www.genome.jp/kegg/). We used KOBAS software to test the statistical enrichment of peak related genes in KEGG pathways. Different peak analysis was based on the fold enrichment of peaks of different experiments. A peak was determined as different peak when the odds ratio between two groups was more than 2. Using the same method, genes associated with different peaks were identified and conduct GO and KEGG enrichment analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA stability assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePUM2 stably knockdown and control MIA PaCa-2 and AsPC-1 cells were treated with Actinomycin D (Sigma-Aldrich) at 5 \u0026mu;g/mL. The time courses of samples with Actinomycin D treatment (0, 4, 8, 12 and 24 h) were used for RNA extraction. RNA was reversed transcription and analyzed by quantitative real-time PCR (qRT-PCR). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDual luciferase reporter assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGV272 luciferase expression system was purchased from Genechem Corporation (China) and performed according to the manufacturer\u0026rsquo;s instructions. ITGA3 reporter plasmid was cloned by inserting the full-length ITGA3-3\u0026prime;UTR and corresponding mutant sequence after the Firefly luciferase (F-luc) coding sequence. Cells seeded in 48-well plate were transfected with 100 ng of F-luc-ITGA3-3\u0026prime;UTR fusion reporter plasmid. After 72h, cells were analyzed with Dual Luciferase system (Yeason). F-luc activity was used to evaluate the binding effect of PUM2 on ITGA3-3\u0026prime;UTR. Renilla Luciferase (R-luc) was used to normalize the transfection efficiency of the reporter plasmid. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe GraphPad Prism version 9.0 (Graphpad, Inc., Chicago, IL) was used for data analysis. Patients\u0026rsquo; survival rates were analyzed using Kaplan\u0026ndash;Meier plots and log-rank tests. The correlations between PUM2 and different clinicopathological parameters were evaluated using Pearson\u0026rsquo;s \u0026chi;2 test. Univariable and multivariable Cox proportional hazard regression models were used to analyze independent prognostic factors. Data are presented as the mean \u0026plusmn; SD of three independent experiments. Results were considered statistically significant for p＜0.05.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePUM2 plays a vital role in PDAC chemoresistance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirst, the sensitivity of AsPC-1/GEM, the GEM-resistant PC strain AsPC-1/GEM and the parental strain AsPC-1, to GEM previously constructed by our research group was verified. (IC50 = 21.67 \u0026mu;M) the sensitivity to GEM was significantly decreased (Fig. 1A). Transcriptome sequencing showed that there were 4551 differentially expressed genes between the GEM-resistant PC strain AsPC-1/GEM and the parental strain AsPC-1 (Fig. 1B). The differentially expressed genes were mainly enriched in the MAPK signaling pathway, tumor-associated glycoprotein tumors, Focal adhesion pathway, Hippo signaling pathway, Rap1 signaling pathway, arginine and proline metabolism pathway, pyrimidine metabolism and other pathways (Supplementary Fig. 1A), among them, the highly expressed genes in AsPC-1/GEM are mainly enriched in MAPK signaling pathway, Hippo signaling pathway, axon guidance pathway, tumor-related microRNA, tight junction, apoptosis, TGF-\u0026beta; pathway, platinum drug resistance, phosphoinositide metabolism, glutathione metabolism and other pathways (Supplementary Fig. 1B).\u003c/p\u003e\n\u003cp\u003eFurthermore, we screened RBPs that may play a key role in the process of GEM resistance in PC through bioinformatics analysis and siRNA library (Fig. 1C). A total of 4551 genes with differential expression in the PC drug-resistant cell line AsPC-1/GEM and the parental strain AsPC-1 were crossed with 1183 RBPs to obtain the PC drug-resistant cell line AsPC-1/GEM and the parental strain There are 145 RBPs with differential expression in AsPC-1. Bioinformatics analysis based on the TGCA database and GTEx database was performed on the above 145 RBPs, and the candidate RBPs were screened by whether the gene had differential expression in PC tissue and normal pancreatic tissue. To 94 RBPs, the candidate RBPs were screened to 23 based on whether there was a significant difference in disease-free survival between patients with high and low expression of this gene (Supplementary Table 1).\u003c/p\u003e\n\u003cp\u003eThen we used the si-RNA library of the above 23 RBPs for subsequent functional screening. First, we reverse transfected the siRNA library into MIA PaCa-2 cells and found that after knocking down RBPs such as PUM2, FLNA, RTN4, DUSP14, and EIF2AK2 for 72 h, the number of cells was significantly reduced compared with the control group (Fig. 1D-E). We selected 7 RBPs that may positively regulate cell proliferation ability for further drug resistance function screening test, and found that after knockdown of PUM2, the sensitivity of MIA PaCa-2 and AsPC-1 to GEM was significantly increased (Fig. 1F-I). In conclusion, among the differentially expressed RBPs (DE-RBPs) associated with PC GEM resistance in this screening, PUM2 is the RBP with the most significant effect on GEM sensitivity and proliferation ability of PC cells.\u003c/p\u003e\n\u003cp\u003eWe performed a separate bioinformatics analysis of PUM2 and found that it was highly expressed in PC tissues compared with normal pancreatic tissues (Fig. 1J, p \u0026lt; 0.05), and DFS was significantly shortened in PC patients with high PUM2 expression (Fig. 1K). In addition, we performed PUM2 immunohistochemical staining on PC tissue chips containing 82 samples, and scored each sample according to the staining intensity and staining area, and determined that 28 patients had high expression of PUM2 and 38 patients had moderate expression of PUM2 16 patients with low expression of PUM2, and then combined with the survival data of patients for survival analysis, it was found that the overall survival of PC patients with high expression of PUM2 was significantly shortened (Fig. 1L). Western Blot verified that the expression of PUM2 in the GEM-resistant PC strain AsPC-1/GEM was significantly higher than that in the parental strain AsPC-1 (Fig. 1M).\u003c/p\u003e\n\u003cp\u003eWe analyzed the correlation between the expression of PUM2 and the clinicopathological characteristics of the patients, and found that the expression of PUM2 had a strong correlation with the degree of tumor cell differentiation (p = 0.031) and T stage (p = 0.029) (Table 1). We defined the postoperative survival period greater than or equal to 24 months as long-term survival, and less than 24 months as short-term survival. Using univariate Cox regression analysis to explore the factors affecting the long-term survival of patients, we found that PUM2 was highly expressed (HR=16.671, 95CI% 4.882-56.922, p＜0.001), N1/2 stage (HR=2.797, 95CI% 1.263-6.197, p = 0.011) were risk factors affecting the long-term survival of patients, and the tumor was located in the body and tail of the pancreas (HR=0.409, 95CI% 0.174-0.964, p = 0.041) is a protective factor affecting the long-term survival of patients. Further multivariate Cox regression analysis was used to explore the factors affecting the long-term survival of patients. It was found that PUM2 was highly expressed (HR=15.280, 95CI% 4.461-52.336, p\u0026lt;0.001) It is an independent risk factor affecting the long-term survival of patients (Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePUM2 promotes proliferation, migration and chemoresistance of PDAC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we performed functional experiments to verify the effect of PUM2 on the malignant biological behavior of PC cells. Using Western Blot and qRT-PCR, we obtained the expression profile of PUM2 in commonly used PC cell lines at the protein and RNA (Fig. 2A-B) levels and found that PUM2 was relatively high in MIA PaCa-2, AsPC-1 Expression, relatively low expression in BxPC-3, PANC-1.\u003c/p\u003e\n\u003cp\u003eWe used lentivirus to construct cell lines with stable knockdown of PUM2 in MIA PaCa-2 and AsPC-1, and constructed cell lines with stable overexpression of PUM2 in BxPC-3 and PANC-1, and performed Western Blot and qRT-PCR. The results showed that the expression level of PUM2 in the two PUM2 stable knockdown cell lines of MIA PaCa-2 PUM2 KD and AsPC-1 PUM2 KD was significantly lower than that of the control cell line (Fig. 2C-D), while BxPC-3 PUM2 OE, PANC The expression level of PUM2 was significantly higher in the two PUM2-overexpressing cell lines -1 PUM2 OE than in the control cell line (Fig. 2E-F).\u003c/p\u003e\n\u003cp\u003eUsing the PC PUM2 stable knockdown and overexpression cell lines constructed above, we performed in vitro functional tests. The results of proliferation experiments showed that knockdown of PUM2 could significantly inhibit the in vitro proliferation of PC cells MIA PaCa-2 and AsPC-1 (Fig. 2J-H), overexpression of PUM2 can promote the in vitro proliferation ability of significant PC cells BxPC-3 and PANC-1 (Fig. 2I-J).\u003c/p\u003e\n\u003cp\u003eGEM cytotoxicity assay showed that knockdown of PUM2 could increase the sensitivity of PC cells MIA PaCa-2 and AsPC-1 to GEM (Fig. 2K-L), while overexpression of PUM2 could reduce the sensitivity of PC cells to GEM (Fig. 2M-N). Transwell cell migration experiments showed that knockdown of PUM2 could significantly inhibit the migration ability of PC cells (Fig. 2O-P, p \u0026lt; 0.001), and overexpression of PUM2 could significantly promote the migration ability of PC cells BxPC-3 and PANC-1 (Fig. 2Q-R, p \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003eFurthermore, we used a mouse subcutaneous xenograft tumor model to verify the effect of PUM2 on PC cell proliferation and GEM sensitivity in vivo (Fig. 3A). We subcutaneously injected two groups of MIA PaCa-2 PUM2 NC cells and MIA PaCa-2 PUM2 KD cells into nude mice, respectively. After 2 weeks of injection, clear tumor formation was observed. GEM (50 mg/kg) was injected intraperitoneally for the second time, and PBS was injected in the other two groups. After 4 weeks of continuous administration, the mice were sacrificed, the transplanted tumors were dissected out and the tumor and volume were measured, and the tumor growth curve was made. The results showed that the PUM2 NC+PBS group had the fastest tumor growth rate, followed by the PUM2 KD+PBS group, while the tumor growth rate was the slowest in the PUM2 KD+GEM group (Fig. 3B). During the 45-day tumorigenic cycle, the subcutaneous tumors of the mice in the PUM2 KD+GEM group had almost no obvious growth, and finally the subcutaneous tumor weight of the mice in this group was significantly lower than that of the mice in the other groups (Fig. 3C-D, p\u0026lt;0.001).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA-seq and RIP-seq jointly reveals RNA regulating function of PUM2 in PDAC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we explored the possible mechanisms by which PUM2 exerts the above-mentioned biological functions in PC, especially in promoting resistance to GEM. First, we performed transcriptome sequencing on the MIA PaCa-2 PUM2 KD stable transfected cell line and its control cell line, MIA PaCa-2 PUM2 NC. Three samples of KD and PUM2 NC can be clustered separately, with small differences within groups and large differences between groups (Supplementary Fig. 2A-B). The sequencing results showed that after knockdown of PUM2, the expression of 2607 genes were significantly up-regulated, the expression of 1966 genes were significantly down-regulated, and the expression of the remaining 22,701 genes had no significant difference between the two groups (Fig. 4A, Supplementary Fig. 2C).\u003c/p\u003e\n\u003cp\u003eFurthermore, we performed GO and KEGG enrichment analysis on the differentially expressed genes after knockdown of PUM2 to find the important pathways and cell biological functions that PUM2 may regulate. In GO enrichment analysis, all differentially expressed genes can be enriched for cell adhesion molecule binding, cell adhesion junction, focal adhesion pathway, chromatin binding, ubiquitin protein ligase binding, cell cycle negative regulation, RNA metabolism process, Important pathways such as ribosome assembly, the analysis of genes whose expression is up-regulated after PUM2 knockdown can be enriched to important pathways such as NADH deoxygenase activity, oxidoreductase activity, mitochondrial protein complex, and ribosomal protein complex biosynthesis. Analysis of genes down-regulated after PUM2 enriched important pathways such as serine/threonine kinase activity, centrosome, proteasome catalytic process, transcriptional repression complex, and regulation of cell-matrix junctions (Fig. 4B, Supplementary Fig. 2D-E).\u003c/p\u003e\n\u003cp\u003eIn the KEGG enrichment analysis, the analysis of all differentially expressed genes can enrich important pathways such as endocrine resistance, longevity regulation pathways, tumor glycoproteins, oxidative phosphorylation, apoptosis, and cell cycle. Gene analysis can enrich important pathways such as N-glycan biosynthesis, cell cycle, spliceosome, oxidative phosphorylation, and thermogenesis. The analysis of genes whose expression is down-regulated after PUM2 knockdown can be enriched in the focal adhesion pathway, NF -\u0026kappa;B signaling pathway, mitophagy, PI3K-AKT signaling pathway, tumor-related choline metabolism, ErbB signaling pathway, longevity regulation pathway, TNF signaling pathway, FoxO signaling pathway, PC-related genes and other important pathways (Supplementary Fig. 3A-C). In addition, GSEA analysis also showed that knockdown of PUM2 significantly decreased the expression of related genes in the focal adhesion pathway in PC cells (Fig. 4C).\u003c/p\u003e\n\u003cp\u003eFurther, we performed RNA co-immunoprecipitation (RIP) experiments of PUM2 protein on MIA PaCa-2 cells, and the RNA products from RIP were exported for RIP-seq. The measured peaks were distributed on all 23 pairs of chromosomes including sex chromosomes (Supplementary Fig. 4A). By matching each peak to the corresponding gene region, it was found that most of the peaks were located in the 3\u0026apos;UTR region and CDS region of the gene (Supplementary Fig. 4B), of which 45.49% of the peaks were matched to the 3\u0026apos;UTR region of the corresponding gene in the genome, and 44.62% of them were matched to the 3\u0026apos;UTR region of the corresponding gene in the genome. The peaks of PUM2 were matched to the CDS regions of the corresponding genes in the genome, and the above two accounted for more than 90% of all peaks (Supplementary Fig. 4C), which was also in line with the previous conclusion that the RNA-binding element PBE of the PUM2 protein mainly binds to the 3\u0026apos;UTR of mRNA. In addition, the enrichment analysis of the genes corresponding to the peaks measured in RIP-seq showed that the focal adhesion pathway, Wnt signaling pathway, Ras signaling pathway, tumor-associated glycoprotein, PI3K-AKT signaling pathway, Notch signaling pathway, mTOR signaling pathway and other important Pathway gene sets were enriched (Fig. 4D).\u003c/p\u003e\n\u003cp\u003eAnalyzing the results of RNA-seq detection, there were many key pathways related to GEM resistance in previous studies in the KEGG enrichment analysis of down-regulated genes after PUM2 knockdown. Therefore, we compared the peaks obtained in RIP-seq with RNA- The genes whose expression was down-regulated after PUM2 knockdown obtained in seq were intersected, and a total of 267 genes were obtained (Fig. 4E). The enrichment analysis of these genes also obtained cell adhesion, chromatin binding, transcriptional repression complex, and transcription factor binding. and other important pathways (Supplementary Fig. 4D). Because focal adhesions and cell adhesion pathways appear in the GO enrichment analysis, KEGG enrichment analysis, RIP-seq peak corresponding gene enrichment analysis and the enrichment analysis results of the two intersection genes of RNA-seq, and the gene set contains ITGA3 gene, that is, the gene with the most obvious difference between the PUM2 knockdown group and the control group in RNA-seq, and previous literature also showed that ITGA3 plays an important role in GEM resistance. Therefore, we selected ITGA3 and its focal adhesion pathway gene set as the possible downstream of PUM2 in regulating GEM resistance in PC for further study (Supplementary Table 2). The abundance of corresponding RNA of typical genes in focal adhesion pathway was significantly higher in PUM2-Ab than that in input (Fig. 4F). We performed bioinformatics analysis based on TCGA and GTEx databases for six candidate genes including ITGA3, ADAM17, ASAP1, FLNA, COL1A2, and COL6A3. The results showed that the above genes were highly expressed in PC tissues, and their expression was significantly positively correlated with the expression level of PUM2. For some genes in the focal adhesion pathway gene set including the above genes (9/27), there was a significant difference in OS between high expression PC patients and low expression patients (p＜0.05); for focal adhesion pathways including the above genes For half of the genes in the pathway gene set (13/27), there was a significant difference in DFS between patients with high expression and low expression (p＜0.05) (Supplementary Table 2, Supplementary Fig. 5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFocal adhesion pathway is regulated integrally by PUM2 at mRNA level in PDAC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to explore whether PUM2 has a regulatory effect on genes related to the focal adhesion pathway gene set such as ITGA3, and the specific mechanism of PUM2 regulating the above genes, we carried out the following experiments to verify. First, we used qRT-PCR to detect the mRNA levels of representative genes (ITGA3, ADAM17, ASAP1, FLNA, COL1A2, COL6A3) in the focal adhesion pathway in the combined analysis of RIP-seq and RNA-seq, and found that knockdown of PUM2 After overexpression of PUM2, the mRNA levels of the above genes in MIA PaCa-2 and AsPC-1 cells were significantly decreased (Fig. 5A-B), and the mRNA levels of the above genes in MIA PaCa-2 and AsPC-1 cells were significantly increased after PUM2 overexpression (Fig. 5C-D), the results confirmed that the mRNA levels of ITGA3, ADAM17, ASAP1, FLNA, COL1A2, and COL6A3 were indeed correlated with the expression levels of PUM2.\u003c/p\u003e\n\u003cp\u003eSecondly, we used RIP-qPCR to verify that PUM2 can bind to the 3\u0026apos;UTR region of the above gene mRNAs, and found that compared with IgG, the PUM2 antibody group could significantly bind to the 3\u0026apos;UTR regions of ITGA3, ADAM17, ASAP1, FLNA, COL1A2 and COL6A3 gene mRNAs (Fig. 5E-H). PUM2 binds to the 3\u0026apos;UTR region of ITGA3 mRNA at the highest level.\u003c/p\u003e\n\u003cp\u003eSubsequently, we used ActD to conduct the RNA stability test of the above genes, and found that after knockdown of PUM2 in both MIA PaCa-2 and AsPC-1 cells, ITGA3, ADAM17, ASAP1, FLNA, COL1A2 and COL6A3 The stability of isogenic mRNA decreased to varying degrees, confirming that PUM2 regulates the mRNA stability of the above-mentioned genes by binding to the 3\u0026apos;UTR region of the above-mentioned genes, and finally regulates the mRNA levels of the above-mentioned genes (Fig. 5I-J).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eITGA3 is a crucial downstream in PUM2 promoting chemoresistance and metastasis of PDAC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eITGA3 is the gene with the most significant difference between the PUM2 knockdown group and the control group in RNA-seq, and its mRNA 3\u0026apos;UTR region is also the most enriched in RIP-qPCR. In addition, a study published in \u003cem\u003eGastroenterology\u0026nbsp;\u003c/em\u003ein 2020 It is shown that ITGA3 can be activated and up-regulated by the upstream ZIP4/ZEB1 pathway in PC cells, thereby activating the downstream JNK pathway and inhibiting the GEM transporter ENT1 on the cell membrane, thereby inducing PC GEM resistance. Therefore, we validated ITGA3 as a key downstream gene of PUM2 to induce GEM resistance in PC cells. First, we performed a dual-luciferase reporter assay to verify the binding of PUM2 to the ITGA3 3\u0026apos;UTR region. By searching the sequence of the ITGA3 3\u0026apos;UTR region, we found that it contains the PBE sequence (-TGTATATA-, Fig. 6A). Based on this, we constructed a wild-type firefly luciferase reporter gene plasmid and a PBE sequence in the 3\u0026apos;UTR region of ITGA3 mRNA, respectively. The PBE sequence mutant firefly luciferase reporter gene plasmid in the 3\u0026apos;UTR region of ITGA3 mRNA was used for the dual luciferase reporter gene assay. The results showed that after knocking down PUM2, the PBE sequence in the 3\u0026apos;UTR region of ITGA3 mRNA was expressed in the middle and downstream of the wild-type plasmid. The degree of expression was significantly reduced (p \u0026lt; 0.01), while the expression degree of the downstream genes of the PBE sequence mutant plasmid in the 3\u0026apos;UTR region of ITGA3 mRNA had no significant change (Fig. 6B). The above experiments proved that PUM2 can bind to the PBE sequence in the 3\u0026apos;UTR region of ITGA3 mRNA.\u003c/p\u003e\n\u003cp\u003eSubsequently, we used Western Blot to verify that after knockdown of PUM2, the protein levels of ITGA3 in MIA PaCa-2 and AsPC-1 cells were significantly decreased, and the protein levels of key proteins AKT and p-AKT in the AKT pathway downstream of the focal adhesion pathway also decreased. significantly decreased (Fig. 6C), while the protein levels of ITGA3, AKT, and p-AKT were significantly increased in BxPC-3 and PANC-1 cells after overexpression of PUM2 (Fig. 6D).\u003c/p\u003e\n\u003cp\u003eFinally, we performed rescue experiments for the function of PUM2 using ITGA3. ITGA3 was overexpressed in MIA PaCa-2 and AsPC-1 cells stably knocked down by PUM2 and verified by Western Blot. The results showed that the cell model was successfully constructed (Fig. 6E). Furthermore, we examined MIA PaCa-2 PUM2 NC cells, MIA PaCa-2 PUM2 KD cells, MIA PaCa-2 PUM2 NC+Vec cells, MIA PaCa-2 PUM2 KD+ITGA3 OE cells and AsPC-1 PUM2 NC cells, AsPC-1 PUM2 KD cells, AsPC-1 PUM2 NC+Vec cells, AsPC-1 PUM2 KD+ITGA3 OE cells, a total of 8 cell lines were sensitive to GEM. It was found that overexpression of ITGA3 could reverse the effect of knockdown of PUM2 in the above cell lines. The effect of increased sensitivity to GEM (Fig. 6F-G). In addition, because the focal adhesion pathway genes including ITGA3 also play an important role in cell adhesion, invasion and metastasis, we tested the migration ability of the above 8 cell lines. The results showed that overexpression of ITGA3 could also reverse the cells after knockdown of PUM2. The effect of reduced mobility (Fig. 6H-I, p\u0026lt;0.001).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEGR1 and PUM2 regulate mutually in PDAC resulting in a cascaded effect\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter the preliminary exploration of the key genes downstream of PUM2 was completed, we turned to further explore the upstream transcription factors that regulate the expression of PUM2. We used the JASPAR database to predict the transcription factors that can bind to the promoter region of PUM2, and then analyzed the predicted possible upstream transcription factors of PUM2 (366), the corresponding genes of mRNAs that could bind to PUM2 in RIP-seq (1535) and RNA- In seq, the down-regulated genes (1966) were crossed after PUM2 knockdown (Fig. 7A), and finally 22 transcription factors were obtained. Bioinformatics analysis showed that among the 22 transcription factors, early growth factor 1 (EGR1) was significantly higher in PC tissues than in normal PC tissues (Figure 7B, p \u0026lt; 0.05), and the expression level of EGR1 in PC tissues It was significantly positively correlated with the expression level of PUM2 (Fig. 7C, R = 0.29, p = 9.7\u0026times;10-5).\u003c/p\u003e\n\u003cp\u003eThen reverse verification was performed, using the CLIP-seq or RIP-seq data in the PARalyzer data set, the FIMO data set and the ENCODE eCLIP data set in the POSTAR website to predict the RNA-binding proteins that may bind to EGR1 mRNA. The results are shown in multiple data sets. Concentratingly, both PUM2 may bind to EGR1 mRNA (Fig. 7D).\u003c/p\u003e\n\u003cp\u003eIn addition, a study published in \u003cem\u003eScience\u003c/em\u003e in 2020 showed that EGR1 plays an important role in pancreatitis-cancer transformation. Therefore, based on the forward and backward prediction results, bioinformatics analysis results and literature reading, we finally selected EGR1 as a candidate upstream transcription factor of PUM2 for follow-up research. Furthermore, we verified the expression regulation relationship between transcription factor EGR1 and RNA-binding protein PUM2 at the RNA and protein levels. The results showed that after knockdown of PUM2 in MIA PaCa-2 and AsPC-1 cells, the expression of EGR1 at both mRNA and protein levels was significantly decreased (Fig. 8A-B), and PUM2 was overexpressed in BxPC-3 and PANC-1 cells After treatment, the expression of EGR1 at both mRNA and protein levels was significantly increased (Fig. 8C-D). Conversely, after knockdown of EGR1 in BxPC-3 cells, the expression of PUM2 at the mRNA and protein levels was also significantly decreased (Fig. 8E-F). After overexpression of EGR1 in PANC-1 cells, the expression of PUM2 at the mRNA and protein levels was significantly reduced Expression was also significantly elevated (Fig. 8G-H).\u003c/p\u003e\n\u003cp\u003eIn terms of the regulatory mechanism of the two, we confirmed by RIP-qPCR that in MIA PaCa-2 and AsPC-1 cells, PUM2 protein can more significantly bind to the 3\u0026apos;UTR region of EGR1 mRNA than IgG (Fig. 8I-J, p＜0.01).\u003c/p\u003e\n\u003cp\u003eFinally, we performed rescue experiments for the function of PUM2 using EGR1. EGR1 was overexpressed in MIA PaCa-2 cells stably knocked down by PUM2 and verified by Western Blot, and the results showed that the cell model was successfully constructed (Fig. 8K). Furthermore, we detected the sensitivity of four cell lines to GEM, including MIA PaCa-2 PUM2 NC cells, MIA PaCa-2 PUM2 KD cells, MIA PaCa-2 PUM2 NC+Vec cells, and MIA PaCa-2 PUM2 KD+EGR1 OE cells. It was found that overexpression of EGR1 could reverse the effect of increased GEM sensitivity caused by knockdown of PUM2 in the above cell lines (Fig. 8L), and overexpression of EGR1 could also reverse the effect of decreased cell migration ability after knockdown of PUM2 (Fig. 8M, p＜0.001).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePC has a high degree of malignancy. Chemotherapy is one of the most important treatment methods for PC. Effective chemotherapy can not only prolong the survival of advanced patients, but also transform unresectable or junctional resectable patients into resectable ones, thereby enabling patients to have a cure. possible. Therefore, how to sensitize PC chemotherapy efficacy and reduce the occurrence of PC chemotherapy resistance has always been one of the key contents in the basic research of PC. At present, the first-line chemotherapy regimens for PC are mainly divided into three categories. One is a combination chemotherapy regimen based on GEM (such as GEM combined with nab-paclitaxel), and the other is based on 5-FU or its derivatives. Combination chemotherapy regimens (such as FOLFIRINOX regimen), and the third is platinum-containing regimens (such as FOLFOX regimen) applied to patients with BRCA mutations. Currently, clinical practice in China mostly adopts the first chemotherapy regimen, that is, GEM-based combination chemotherapy regimen. Therefore, in-depth study of the GEM resistance mechanism of PC and the search for targeted therapy to sensitize GEM are effective means to improve the efficacy of PC chemotherapy. RBP is an essential protein in the process of intracellular RNA metabolism. RNA is a key part of the central dogma. Therefore, RBP that regulates its various metabolic processes is also of great significance to the regulation of cellular functions. Although some studies on RBP in PC have been reported, there is still a lack of systematic research on which RBP plays a key role in GEM resistance of PC. Therefore, this subject has carried out in-depth research from exploration to verification, from in vivo to in vitro, and from function to mechanism, with the scientific question of exploring the functions and specific mechanisms of key RBPs in GEM resistance of PC.\u003c/p\u003e \u003cp\u003eBased on the transcriptome sequencing data of GEM-resistant PC strains and parental strains, and PC gene expression and survival data from the TCGA database, we first screened out candidate RBPs that may be related to GEM resistance in PC, and then constructed siRNA for proliferation and GEM The cytotoxicity test showed that PUM2 was the RBP with the strongest correlation with GEM resistance in PC. Based on the analysis of PUM2 immunohistochemical staining and clinicopathological characteristics of PC tissue chips, it was confirmed that the high expression of PUM2 is closely related to the poor prognosis of PC.\u003c/p\u003e \u003cp\u003ePUM2, one of the mammalian Pumilio proteins, is a member of the PUF family of sequence-specific RNA-binding proteins [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Pumilio protein was first discovered because of its important role in Drosophila embryonic development [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and has since been widely regarded as a typical post-transcriptional regulator. There are two kinds of Pumilio proteins in the human body, namely PUM2 and PUM1, which have 76% homology and 30% homology with the Pumilio protein in Drosophila. The structural feature of PUM2 is that it contains 1 interspecies homology. The highly conserved Pumilio homology domain (PUM-HD) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] also contains a conserved sequence that can bind to mRNA, the Pumilio recognition element (PRE) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In recent years, the function of PUM2 in tumorigenesis and development has been gradually recognized [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. For example, PUM2 is highly expressed in acute myeloid leukemia tumor cells, which can affect the proliferation, cell cycle regulation and apoptosis of hematopoietic stem cells. In terms of mechanism, PUM2 can use PRE to bind to FOXP1 mRNA to activate FOXP1 expression, and ultimately inhibit cell cycle inhibitory factors, for example, the expression of CDKN2B [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]; another example, PUM1 in the PUM family in seminoma functions as a post-transcriptional repressor, which can inhibit the expression of SPIN1, an important regulatory protein of meiosis at the post-transcriptional level, thereby inhibiting spermatogenesis. Proliferation of primary cell tumors and promote apoptosis [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In addition to mRNA, PUM proteins can also affect the occurrence and development of tumors by regulating the functions of non-coding RNAs such as miRNA and lncRNA [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. For example, in glioblastoma, CDKN1B mRNA can be bound by miR-221 and miR-222, and PUM2 can cause local conformational rearrangement of CDKN1B mRNA by binding to the 3'UTR region, which in turn leads to the exposure of complementary miRNA binding sites and synergy Enhance the inhibitory effect of miRNA on CDKN1B expression [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, to explore the mechanism of PUM2 promoting PC resistance to GEM, we combined RIP-seq and RNA-seq to find that PUM2 can bind to the 3'UTR region of focal adhesion-related gene mRNAs such as ITGA3, thereby improving mRNA stability The expression levels of focal adhesion-related genes were up-regulated, and the regulatory effect of PUM2 on ITGA3 was verified step by step by RIP-qPCR, RNA stability experiments, dual luciferase reporter experiments and functional rescue experiments. The post-transcriptional regulation of PUM2 on downstream target mRNAs is mostly post-transcriptional inhibition [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The 5'7-methylated guanylate cap (5'cap) and 3 The 'polyadenylation tail is the main structure that prevents mRNA from being degraded [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], while PUM2 binds to the 3' polyadenylation tail of mRNA and recruits the CNOT family with deadenylase to induce mRNA degradation[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] 44, 45]. In addition, the N-terminus of PUM2 also has a conserved repression domain (RD), which is currently believed to bind to the 5' cap structure of mRNA to produce a \"decapping\" effect to accelerate mRNA degradation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. At the same time, the Pum-HD structure in PUM2 can inhibit the role of PABP in promoting transcription initiation without dissociating PABP protein from mRNA, but the specific mechanism is currently unknown [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In addition to post-transcriptional inhibition, PUM2 can also play a post-transcriptional activation effect on some genes. For example, in the aforementioned acute myeloid leukemia, PUM2 can play a post-transcriptional activation role by binding to the 3'UTR region of FOXP1 mRNA and upregulate its expression. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Therefore, PUM2 plays different post-transcriptional regulatory roles for different genes. Similarly, iron response element protein (IREP) plays different post-transcriptional regulatory roles depending on the sequence of the binding site. Cytoplasmic poly-adenylation element-binding protein (CPEB) can also exert different post-transcriptional regulatory roles according to different developmental cues [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. This study confirms that PUM2 can up-regulate the expression level of focal adhesion-related genes by inhibiting the degradation rate of focal adhesion-related genes in PC cells, but the specific mechanism of the deeper PUM2-mediated post-transcriptional activation has not been reported in previous studies. In the follow-up study, we can conduct Co-IP experiment and RNA pull down experiment on PUM2 protein in PC, and even try to conduct Co-IP experiment of PUM2 protein on the protein product of RNA pull down experiment, to explore whether PUM2 can interact with PUM2. Other post-transcriptional regulators, such as proteins that promote 3' polyadenylation, form a complex to play a role in post-transcriptional activation; or whether PUM2 competes with important post-transcriptional repression regulators of focal adhesion-related gene mRNAs Inhibition and post-transcriptional activation.\u003c/p\u003e \u003cp\u003eThe key downstream gene of PUM2 in this study is ITGA3, which encodes the α3 subunit of the integrin family. Integrin proteins are a class of ubiquitous heterodimeric transmembrane glycoprotein receptors, mainly present on mammalian cell membranes as signal transduction proteins [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Previous studies have shown that the integrin family can promote PC metastasis and resistance to chemotherapy. For example, integrin α2β1 increases the resistance of PC cells to 5-FU by upregulating the expression of Bcl-2 [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]; another example, ITGB1 can promote GEM resistance of PC by activating Cdc42 [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The integrin α3 subunit often non-covalently binds to the β1 subunit to form the integrin α3β1 receptor anchored on the cell membrane. A study published in \"Gastroenterology\" in 2020 showed that the zinc finger protein ZIP4 promotes the transcription factor ZEB1 by activating the STAT pathway The expression of integrin α3β1 on the cell membrane can then activate the intracellular JNK pathway, thereby inhibiting the expression of the GEM transporter ENT1 on the cell membrane, and finally promoting the GEM resistance of PC cells function [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Therefore, the up-regulation of ITGA3 expression caused by the high expression of PUM2 in this study can be clearly regarded as one of the factors explaining the GEM resistance of PC induced by PUM2. In recent years, many drugs targeting the integrin family have appeared. Although there is still no FDA-approved integrin family-targeting drug for tumor therapy, some of them have been used in tumor-related clinical trials [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. For example, GLPG-187, which targets integrins containing the αv subunit, is being tested for the treatment of various solid tumors, and Cilengitide, which targets integrins αvβ3 and αvβ5, is being tested for the treatment of glioblastoma, OS2966, which targets β1 subunit-containing integrins, was tested for the treatment of glioma [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. At present, it may be difficult to develop small molecule inhibitors or monoclonal antibodies targeting PUM2 from scratch. Instead, we can try to inhibit PUM2 to play an important downstream ITGA3 in promoting GEM resistance in PC to achieve GEM sensitization. There is also no specific drug targeting integrins containing α3 subunit, but in the follow-up experiments, it can be considered to use OS2966 targeting integrins containing β1 subunit to inhibit the function of integrin α3β1 and achieve GEM sensitization effect. In addition, combined with the rapid development of artificial intelligence and deep learning technology this year, we will consider applying deep learning methods in the future to find drugs that may inhibit PUM2 or ITGA3 among FDA-approved drugs, that is, \"Drugs targeting PUM2 or ITGA3\" repropose\u0026rdquo;, which will be subsequently verified by cell and animal experiments, in order to transform this topic into an effective study that can truly sensitize PC GEM chemotherapy.\u003c/p\u003e \u003cp\u003eIn addition to the focal adhesion-related gene set including ITGA3, many pathways were enriched in RIP-seq and RNA-seq in this study, and these pathways may also be involved in PUM2-induced PC GEM resistance, and even PUM2-induced PC It plays an important role in other malignant biological behaviors of cancer. For example, NF-κB signaling pathway, FoxO signaling pathway enriched in RNA-seq, Wnt signaling pathway, Notch signaling pathway, and mTOR signaling pathway enriched in RIP-seq have all been reported to be related to the occurrence and development of PC, and even PC GEM resistance is closely related [\u003cspan additionalcitationids=\"CR56 CR57 CR58\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. It is worth noting that in the two functional enrichment analysis of RNA-seq in this study and the gene enrichment results of up-regulated expression after PUM2 knockdown, oxidative phosphorylation-related gene sets, NADH activity-related gene sets, and oxidoreductases appeared. Activity-related gene set, located in mitochondrial inner membrane protein gene set. Specific to specific genes, after knocking down PUM2 in this study, mitochondrial ribosomal proteins (mitochondrial ribosomal proteins, MRPs), isocitrate dehydrogenase (IDH2, IDH3G), mitochondrial membrane proteins (VDAC1, TOMM40, CHCHD1, CHCHD2, CHCHD10 etc.) expression was significantly increased, suggesting that after knockdown of PUM2, the aerobic respiration efficiency of PC cells may increase, and it can be speculated that the aerobic respiration efficiency is low when PUM2 is highly expressed, and the energy supply of cells may mainly depend on anaerobic glycolysis. This is consistent with the conclusion of previous studies that anaerobic glycolysis can promote GEM resistance in PC [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. By reading the literature, we were pleasantly surprised to find that the phenomenon that PUM2 promotes anaerobic glycolysis is not our speculation. A study published in \"Molecular Cell\" in 2019 suggests that PUM2 is an RBP that plays an important role in the regulation of aging. The high expression of PUM2 in aging cells can downregulate the expression of its downstream mitochondrial fission factor (Mff). Decreased fission ability leads to mitochondrial dysfunction, whereas knockdown of PUM2 can enhance mitochondrial function. The Seahorse results in this paper also confirmed that in the case of knockdown of PUM2, the rate of cellular aerobic respiration was significantly reduced [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Therefore, we intend to focus on mitochondrial function and oxidative respiration as an entry point to study whether PUM2 plays a more important and extensive biological function in PC cells by regulating mitochondrial function and leading to cellular metabolic reprogramming.\u003c/p\u003e \u003cp\u003eIt should be noted that only taking the perspective of PUM2 regulating the downstream mRNA metabolism level as an entry point may still not fully explain and solve the major problem of GEM resistance in PC. Key cues for regulatory function. In 2016, a study published by S. Lee et al. in \"Cell\" showed that noncoding RNA activated by DNA damage (NORAD), which is closely related to DNA damage, contains 15 PBE sequences, and the high expression of NORAD can be expressed in the cytoplasm. The local enrichment of PUM2 in the interior leads to the phenomenon of phase separation, which promotes DNA damage repair and the expression level of cell cycle-related genes, while the inactivation of NORAD leads to a decrease in the level of NORAD-PUM2 phase separation, which can eventually lead to intracellular staining through chromosomal abnormalities and mitotic abnormalities. It is qualitatively unstable, which is of great significance for tumorigenesis [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Although the group's follow-up study suggested that NORAD is more distributed in the nucleus under stress, and that the RNA-binding protein RBMX rather than PUM2 plays a role in NORAD-mediated regulation of chromatin stability, other studies have suggested the important role of PUM2 in this process [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Two studies published in \u003cem\u003eeLife\u003c/em\u003e confirmed from in vitro and in vivo conditions that NORAD in the cytoplasm can function as a PUM2 sponge independent of stress state, thereby regulating genome stability and mitotic processes, and PUM2 overexpression can simulate A phenotype of genomic instability and mitochondrial dysfunction resulting from NORAD deletion [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Taken together, the above studies are not only in line with the high expression of PUM2 may lead to mitochondrial dysfunction, but also suggest that high expression of PUM2 may lead to serious consequences of intracellular genome instability and mitotic dysregulation. Therefore, in follow-up studies, we will also focus on whether the expression level of PUM2 in PC cells is related to genome stability, and whether PUM2 may act as a key driver gene to cause PC through the chromatin instability pathway.\u003c/p\u003e \u003cp\u003eIn the last part of this study, we also explored the transcription factors that may interact with PUM2, and found that PUM2 and EGR1 can function as RNA-binding proteins and transcription factors, respectively, to promote the expression of each other. Therefore, the two are in PC. A cascade amplification effect is formed in the occurrence of GEM resistance. In this study, the RIP-qPCR experiment was used to verify the direct binding effect of PUM2 on EGR1 mRNA, and the ChIP-qPCR experiment can be used to further verify the binding effect of EGR1 and the PUM2 gene promoter region. EGR1, a member of the early growth response factor (EGR) family, can bind to the EGR1-binding sequence (EBS) in the promoter region of downstream genes, CC(A/T)6GG, Binding to promote the expression of downstream genes [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Growth factors, tumor necrosis factors, inflammatory factors, radiotherapy, and reactive oxygen species can induce increased expression of EGR1 [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. EGR1 is highly expressed in gliomas, lung cancers, gastrointestinal tumors, and melanomas. EGR1 can play a role in promoting tumor cell proliferation, invasion and metastasis [\u003cspan additionalcitationids=\"CR71 CR72\" citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. For example, EGR1, whose expression is up-regulated by the activated MAPK pathway in prostate cancer cells, can directly bind to the promoter region of the Cyclin D1 gene, thereby up-regulating the expression of Cyclin D1 and promoting the proliferation of prostate cancer cells [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]; for another example, in hepatocellular carcinoma,, EGR1 can up-regulate SNAIL and SLUG, two important regulators of epithelial-mesenchymal transition (EMT), by activating transcription, thereby promoting the invasion and metastasis ability of liver cancer cells [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e] [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. In PC, a 2021 study on pancreatic adenocarcinoma transformation in \u003cem\u003eScience\u003c/em\u003e found extensive changes in chromatin openness in repaired pancreatic ductal epithelial cells after pancreatitis remission. Changing the regional peaks can enrich several key transcription factors, including EGR1. In the presence of KRAS mutation, EGR1 wild-type mice have a significantly higher incidence of PC and better survival than EGR1-null mice. shortening, and the activation of EGR1 in this inflammatory-cancer transformation may be mediated by IL-6 [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. This shows that EGR1 plays an important role in the development of PC. Combined with our findings, we can continue to explore other functions of EGR1 in PC progression and GEM resistance.\u003c/p\u003e \u003cp\u003eTaking a deeper look at the problem of GEM resistance in PC, the failed GEM sensitization clinical trial in PC suggests that a single pathway that induces GEM resistance may not be enough to reverse resistance, and there may be more advanced PC resistance cells. From an epigenetic perspective, changes in chromatin structure and openness are important factors in regulating the transcription of a wide range of genes. The chromatin structure needs to expose the DNA sequence during replication and transcription. This exposed region is the chromatin open region. This region can be combined by transcription factors and other regulatory elements, so the degree of chromatin openness determines the transcription of cellular genes. activity level [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. In recent years, following histone modification and DNA modification, chromatin openness has become another key area in epigenetic research. ATAC-seq (Assay for Targeting Accessible-Chromatin with high-throughout sequencing) is used to study staining, the preferred method for qualitative accessibility and openness [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. In October 2018, a pan-cancer chromatin openness study based on the ATAC-seq method published in \u003cem\u003eScience\u003c/em\u003e showed that the chromatin openness of tumor cells was significantly different from that of normal cells [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Published in \"Nature\" in February 2021. The research on the mechanism of PC based on single-cell ATAC-seq technology shows that tissue damage and KRAS mutation can induce changes in the degree of chromatin opening of pancreatic cells from an epigenetic perspective, which promotes acinar Ductal metaplasia (ADM) continues to progress to precancerous lesions and even PC [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. In recent years, more and more important studies have shown that changes in the degree of chromatin openness are closely related to the treatment sensitivity of various tumors. For example, in BRCA-mutated tumor cells, if the chromatin remodeling protein ALC1 is lost, it will lead to staining In differentiated thyroid cancers with BrafV600E mutations, the loss of the SWI/SNF chromatin remodeling complex induces a decrease in the degree of chromatin openness, resulting in increased sensitivity to MAPK inhibitors. The SY242CS mutation increases chromatin openness in metastatic ER\u0026thinsp;+\u0026thinsp;breast cancer, leading to resistance to aromatase inhibitor therapy in ER\u0026thinsp;+\u0026thinsp;breast cancer patients [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. So far, there is no relevant research report on how the degree of chromatin openness changes in the process of GEM drug resistance in PC, and what mechanism is used to induce drug resistance. Therefore, further research on PC from the epigenetic perspective of chromatin openness is required. GEM resistance mechanism and sensitizing GEM efficacy may have better prospects and hope.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, we have confirmed that PUM2 is a key RNA-binding protein in inducing GEM resistance in PC through in vitro and in vivo experiments and explored the regulatory role of PUM2 on downstream ITGA3 and other focal adhesion-related genes in PC, and its reciprocal regulation relationship with transcription factor EGR1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). It provides clues for the follow-up further study of other biological functions of PUM2 in the occurrence and development of PC, and also provides a new theoretical basis for understanding the GEM resistance of PC and provides a new idea for the treatment effect of sensitizing GEM in PC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWWB and ZYP designed and supervised the study. ZBB, QC and LZR performed the experiments and collected the data. WYY and LTY contributed to the in vivo experiments. YXY and ZYT contributed to data interpretation. ZBB, QC and LZR performed data analyses. ZBB interpreted the data and wrote the manuscript. All authors read and approved the final manuscript.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWWB received support from the National Natural Science Foundation of China (No. 82173074), CAMS Innovation Fund for Medical Sciences (No. 2021-I2M-1-002), National High Level Hospital Clinical Research Funding (No.2022-PUMCH-B-004) and National Multidisciplinary Cooperative Diagnosis and Treatment Capacity Building Project for Major Diseases. ZYP received support from the CAMS Innovation Fund for Medical Sciences (No. 2021-I2M-1-002) and National High Level Hospital Clinical Research Funding (No.2022-PUMCH-D-001). QC received support from the Fundamental Research Fund for the Central Universities (No. 3332022114). The grants supported this study just financially and had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article/ Supplementary Material. Further inquiries can be directed to the corresponding author.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal procedures were performed in accordance with the European Community guidelines and were approved by the Institutional Animal Care Committee of \u0026ldquo;G. d\u0026rsquo;Annunzio\u0026rdquo; University and by the Italian Ministry of Health (Authorization n. 892/2018-PR). The study was reviewed and approved by\u003c/p\u003e\n\u003cp\u003ethe Ethical Committee of the \u0026ldquo;G. d\u0026rsquo;Annunzio\u0026rdquo; University and Local Health Authority n.2 Lanciano Vasto Chieti, Italy (PROT. 1945/09 COET of 14/07/2009, amended in 2012). The study was performed, after written informed consent from patients, in accordance with the principles outlined in the Declaration of Helsinki.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSIEGEL R L, MILLER K D, FUCHS H E, et al. Cancer statistics, 2022 [J]. CA Cancer J Clin, 2022, 72(1): 7-33.\u003c/li\u003e\n\u003cli\u003eMIZRAHI J D, SURANA R, VALLE J W, et al. Pancreatic cancer [J]. Lancet, 2020, 395(10242): 2008-20.\u003c/li\u003e\n\u003cli\u003ePUSCEDDU S, GHIDINI M, TORCHIO M, et al. Comparative Effectiveness of Gemcitabine plus Nab-Paclitaxel and FOLFIRINOX in the First-Line Setting of Metastatic Pancreatic Cancer: A Systematic Review and Meta-Analysis [J]. Cancers (Basel), 2019, 11(4).\u003c/li\u003e\n\u003cli\u003eSAIF M W, LEE Y, KIM R. Harnessing gemcitabine metabolism: a step towards personalized medicine for pancreatic cancer [J]. Ther Adv Med Oncol, 2012, 4(6): 341-6.\u003c/li\u003e\n\u003cli\u003eCONROY T, HAMMEL P, HEBBAR M, et al. FOLFIRINOX or Gemcitabine as Adjuvant Therapy for Pancreatic Cancer [J]. N Engl J Med, 2018, 379(25): 2395-406.\u003c/li\u003e\n\u003cli\u003eBINENBAUM Y, NA\u0026apos;ARA S, GIL Z. Gemcitabine resistance in pancreatic ductal adenocarcinoma [J]. Drug Resist Updat, 2015, 23: 55-68.\u003c/li\u003e\n\u003cli\u003eW\u0026Ouml;RMANN S M, SONG L, AI J, et al. Loss of P53 Function Activates JAK2-STAT3 Signaling to Promote Pancreatic Tumor Growth, Stroma Modification, and Gemcitabine Resistance in Mice and Is Associated With Patient Survival [J]. Gastroenterology, 2016, 151(1): 180-93.e12.\u003c/li\u003e\n\u003cli\u003eYE Z, HU Q, ZHUO Q, et al. Abrogation of ARF6 promotes RSL3-induced ferroptosis and mitigates gemcitabine resistance in pancreatic cancer cells [J]. Am J Cancer Res, 2020, 10(4): 1182-93.\u003c/li\u003e\n\u003cli\u003eROSS K C, ANDREWS A J, MARION C D, et al. Identification of the Serine Biosynthesis Pathway as a Critical Component of BRAF Inhibitor Resistance of Melanoma, Pancreatic, and Non-Small Cell Lung Cancer Cells [J]. Mol Cancer Ther, 2017, 16(8): 1596-609.\u003c/li\u003e\n\u003cli\u003eMENG Q, LIANG C, HUA J, et al. A miR-146a-5p/TRAF6/NF-kB p65 axis regulates pancreatic cancer chemoresistance: functional validation and clinical significance [J]. Theranostics, 2020, 10(9): 3967-79.\u003c/li\u003e\n\u003cli\u003eCATENACCI D V, JUNTTILA M R, KARRISON T, et al. Randomized Phase Ib/II Study of Gemcitabine Plus Placebo or Vismodegib, a Hedgehog Pathway Inhibitor, in Patients With Metastatic Pancreatic Cancer [J]. J Clin Oncol, 2015, 33(36): 4284-92.\u003c/li\u003e\n\u003cli\u003eHARTWIG W, STROBEL O, HINZ U, et al. CA19-9 in potentially resectable pancreatic cancer: perspective to adjust surgical and perioperative therapy [J]. Ann Surg Oncol, 2013, 20(7): 2188-96.\u003c/li\u003e\n\u003cli\u003eIOKA T, OKUSAKA T, OHKAWA S, et al. Efficacy and safety of axitinib in combination with gemcitabine in advanced pancreatic cancer: subgroup analyses by region, including Japan, from the global randomized Phase III trial [J]. Jpn J Clin Oncol, 2015, 45(5): 439-48.\u003c/li\u003e\n\u003cli\u003eGLISOVIC T, BACHORIK J L, YONG J, et al. RNA-binding proteins and post-transcriptional gene regulation [J]. FEBS Lett, 2008, 582(14): 1977-86.\u003c/li\u003e\n\u003cli\u003eMOORE K S, VON LINDERN M. RNA Binding Proteins and Regulation of mRNA Translation in Erythropoiesis [J]. Front Physiol, 2018, 9: 910.\u003c/li\u003e\n\u003cli\u003eDICTENBERG J B, SWANGER S A, ANTAR L N, et al. A direct role for FMRP in activity-dependent dendritic mRNA transport links filopodial-spine morphogenesis to fragile X syndrome [J]. Dev Cell, 2008, 14(6): 926-39.\u003c/li\u003e\n\u003cli\u003eLEWIS K, VALANEJAD L, CAST A, et al. RNA Binding Protein CUGBP1 Inhibits Liver Cancer in a Phosphorylation-Dependent Manner [J]. Mol Cell Biol, 2017, 37(16).\u003c/li\u003e\n\u003cli\u003eXU W P, YI M, LI Q Q, et al. Perturbation of MicroRNA-370/Lin-28 homolog A/nuclear factor kappa B regulatory circuit contributes to the development of hepatocellular carcinoma [J]. Hepatology, 2013, 58(6): 1977-91.\u003c/li\u003e\n\u003cli\u003eYE L, LIN S T, MI Y S, et al. Overexpression of LARP1 predicts poor prognosis of colorectal cancer and is expected to be a potential therapeutic target [J]. Tumour Biol, 2016, 37(11): 14585-94.\u003c/li\u003e\n\u003cli\u003eFAGOONEE S, PICCO G, ORSO F, et al. The RNA-binding protein ESRP1 promotes human colorectal cancer progression [J]. Oncotarget, 2017, 8(6): 10007-24.\u003c/li\u003e\n\u003cli\u003eBISH R, VOGEL C. RNA binding protein-mediated post-transcriptional gene regulation in medulloblastoma [J]. Mol Cells, 2014, 37(5): 357-64.\u003c/li\u003e\n\u003cli\u003eBRODY J R, DIXON D A. Complex HuR function in pancreatic cancer cells [J]. Wiley Interdiscip Rev RNA, 2018, 9(3): e1469.\u003c/li\u003e\n\u003cli\u003eCOSTANTINO C L, WITKIEWICZ A K, KUWANO Y, et al. The role of HuR in gemcitabine efficacy in pancreatic cancer: HuR Up-regulates the expression of the gemcitabine metabolizing enzyme deoxycytidine kinase [J]. Cancer Res, 2009, 69(11): 4567-72.\u003c/li\u003e\n\u003cli\u003eJIMBO M, BLANCO F F, HUANG Y H, et al. Targeting the mRNA-binding protein HuR impairs malignant characteristics of pancreatic ductal adenocarcinoma cells [J]. Oncotarget, 2015, 6(29): 27312-31.\u003c/li\u003e\n\u003cli\u003eLAL S, CHEUNG E C, ZAREI M, et al. CRISPR Knockout of the HuR Gene Causes a Xenograft Lethal Phenotype [J]. Mol Cancer Res, 2017, 15(6): 696-707.\u003c/li\u003e\n\u003cli\u003eBURKHART R A, PINEDA D M, CHAND S N, et al. HuR is a post-transcriptional regulator of core metabolic enzymes in pancreatic cancer [J]. RNA Biol, 2013, 10(8): 1312-23.\u003c/li\u003e\n\u003cli\u003eZAREI M, LAL S, PARKER S J, et al. Posttranscriptional Upregulation of IDH1 by HuR Establishes a Powerful Survival Phenotype in Pancreatic Cancer Cells [J]. Cancer Res, 2017, 77(16): 4460-71.\u003c/li\u003e\n\u003cli\u003eBLANCO F F, JIMBO M, WULFKUHLE J, et al. The mRNA-binding protein HuR promotes hypoxia-induced chemoresistance through posttranscriptional regulation of the proto-oncogene PIM1 in pancreatic cancer cells [J]. Oncogene, 2016, 35(19): 2529-41.\u003c/li\u003e\n\u003cli\u003eLI C, JIANG J Y, WANG J M, et al. BAG3 regulates stability of IL-8 mRNA via interplay between HuR and miR-4312 in PDACs [J]. Cell Death Dis, 2018, 9(9): 863.\u003c/li\u003e\n\u003cli\u003eGOLDSTROHM A C, HALL T M T, MCKENNEY K M. Post-transcriptional Regulatory Functions of Mammalian Pumilio Proteins [J]. Trends Genet, 2018, 34(12): 972-90.\u003c/li\u003e\n\u003cli\u003eN\u0026Uuml;SSLEIN-VOLHARD C, FROHNH\u0026Ouml;FER H G, LEHMANN R. Determination of anteroposterior polarity in Drosophila [J]. Science, 1987, 238(4834): 1675-81.\u003c/li\u003e\n\u003cli\u003eBARKER D D, WANG C, MOORE J, et al. Pumilio is essential for function but not for distribution of the Drosophila abdominal determinant Nanos [J]. Genes Dev, 1992, 6(12a): 2312-26.\u003c/li\u003e\n\u003cli\u003eZAMORE P D, WILLIAMSON J R, LEHMANN R. The Pumilio protein binds RNA through a conserved domain that defines a new class of RNA-binding proteins [J]. Rna, 1997, 3(12): 1421-33.\u003c/li\u003e\n\u003cli\u003eMURATA Y, WHARTON R P. Binding of pumilio to maternal hunchback mRNA is required for posterior patterning in Drosophila embryos [J]. Cell, 1995, 80(5): 747-56.\u003c/li\u003e\n\u003cli\u003eZAMORE P D, BARTEL D P, LEHMANN R, et al. The PUMILIO-RNA interaction: a single RNA-binding domain monomer recognizes a bipartite target sequence [J]. Biochemistry, 1999, 38(2): 596-604.\u003c/li\u003e\n\u003cli\u003eSMIALEK M J, ILASLAN E, SAJEK M P, et al. Role of PUM RNA-Binding Proteins in Cancer [J]. Cancers (Basel), 2021, 13(1).\u003c/li\u003e\n\u003cli\u003eNAUDIN C, HATTABI A, MICHELET F, et al. PUMILIO/FOXP1 signaling drives expansion of hematopoietic stem/progenitor and leukemia cells [J]. Blood, 2017, 129(18): 2493-506.\u003c/li\u003e\n\u003cli\u003eJANECKI D M, SAJEK M, SMIALEK M J, et al. SPIN1 is a proto-oncogene and SPIN3 is a tumor suppressor in human seminoma [J]. Oncotarget, 2018, 9(65): 32466-77.\u003c/li\u003e\n\u003cli\u003eGALGANO A, FORRER M, JASKIEWICZ L, et al. Comparative analysis of mRNA targets for human PUF-family proteins suggests extensive interaction with the miRNA regulatory system [J]. PLoS One, 2008, 3(9): e3164.\u003c/li\u003e\n\u003cli\u003eZHANG C Z, ZHANG J X, ZHANG A L, et al. MiR-221 and miR-222 target PUMA to induce cell survival in glioblastoma [J]. Mol Cancer, 2010, 9: 229.\u003c/li\u003e\n\u003cli\u003eKEDDE M, VAN KOUWENHOVE M, ZWART W, et al. A Pumilio-induced RNA structure switch in p27-3\u0026apos; UTR controls miR-221 and miR-222 accessibility [J]. Nat Cell Biol, 2010, 12(10): 1014-20.\u003c/li\u003e\n\u003cli\u003eVAN ETTEN J, SCHAGAT T L, HRIT J, et al. Human Pumilio proteins recruit multiple deadenylases to efficiently repress messenger RNAs [J]. J Biol Chem, 2012, 287(43): 36370-83.\u003c/li\u003e\n\u003cli\u003eJACKSON R J, HELLEN C U, PESTOVA T V. The mechanism of eukaryotic translation initiation and principles of its regulation [J]. Nat Rev Mol Cell Biol, 2010, 11(2): 113-27.\u003c/li\u003e\n\u003cli\u003eWEIDMANN C A, RAYNARD N A, BLEWETT N H, et al. The RNA binding domain of Pumilio antagonizes poly-adenosine binding protein and accelerates deadenylation [J]. Rna, 2014, 20(8): 1298-319.\u003c/li\u003e\n\u003cli\u003eJOLY W, CHARTIER A, ROJAS-RIOS P, et al. The CCR4 deadenylase acts with Nanos and Pumilio in the fine-tuning of Mei-P26 expression to promote germline stem cell self-renewal [J]. Stem Cell Reports, 2013, 1(5): 411-24.\u003c/li\u003e\n\u003cli\u003eWEIDMANN C A, GOLDSTROHM A C. Drosophila Pumilio protein contains multiple autonomous repression domains that regulate mRNAs independently of Nanos and brain tumor [J]. Mol Cell Biol, 2012, 32(2): 527-40.\u003c/li\u003e\n\u003cli\u003eCHRITTON J J, WICKENS M. A role for the poly(A)-binding protein Pab1p in PUF protein-mediated repression [J]. J Biol Chem, 2011, 286(38): 33268-78.\u003c/li\u003e\n\u003cli\u003eHENTZE M W, MUCKENTHALER M U, ANDREWS N C. Balancing acts: molecular control of mammalian iron metabolism [J]. Cell, 2004, 117(3): 285-97.\u003c/li\u003e\n\u003cli\u003eIVSHINA M, LASKO P, RICHTER J D. Cytoplasmic polyadenylation element binding proteins in development, health, and disease [J]. Annu Rev Cell Dev Biol, 2014, 30: 393-415.\u003c/li\u003e\n\u003cli\u003eHYNES R O. Integrins: a family of cell surface receptors [J]. Cell, 1987, 48(4): 549-54.\u003c/li\u003e\n\u003cli\u003eAOUDJIT F, VUORI K. Integrin signaling in cancer cell survival and chemoresistance [J]. Chemother Res Pract, 2012, 2012: 283181.\u003c/li\u003e\n\u003cli\u003eYANG D, TANG Y, FU H, et al. Integrin \u0026beta;1 promotes gemcitabine resistance in pancreatic cancer through Cdc42 activation of PI3K p110\u0026beta; signaling [J]. Biochem Biophys Res Commun, 2018, 505(1): 215-21.\u003c/li\u003e\n\u003cli\u003eLIU M, ZHANG Y, YANG J, et al. ZIP4 Increases Expression of Transcription Factor ZEB1 to Promote Integrin \u0026alpha;3\u0026beta;1 Signaling and Inhibit Expression of the Gemcitabine Transporter ENT1 in Pancreatic Cancer Cells [J]. Gastroenterology, 2020, 158(3): 679-92.e1.\u003c/li\u003e\n\u003cli\u003eSLACK R J, MACDONALD S J F, ROPER J A, et al. Emerging therapeutic opportunities for integrin inhibitors [J]. Nat Rev Drug Discov, 2022, 21(1): 60-78.\u003c/li\u003e\n\u003cli\u003eWANG L, ZHOU W, ZHONG Y, et al. Overexpression of G protein-coupled receptor GPR87 promotes pancreatic cancer aggressiveness and activates NF-\u0026kappa;B signaling pathway [J]. Mol Cancer, 2017, 16(1): 61.\u003c/li\u003e\n\u003cli\u003ePRAMANIK K C, FOFARIA N M, GUPTA P, et al. CBP-mediated FOXO-1 acetylation inhibits pancreatic tumor growth by targeting SirT [J]. Mol Cancer Ther, 2014, 13(3): 687-98.\u003c/li\u003e\n\u003cli\u003eZHOU C, YI C, YI Y, et al. LncRNA PVT1 promotes gemcitabine resistance of pancreatic cancer via activating Wnt/\u0026beta;-catenin and autophagy pathway through modulating the miR-619-5p/Pygo2 and miR-619-5p/ATG14 axes [J]. Mol Cancer, 2020, 19(1): 118.\u003c/li\u003e\n\u003cli\u003eYABUUCHI S, PAI S G, CAMPBELL N R, et al. Notch signaling pathway targeted therapy suppresses tumor progression and metastatic spread in pancreatic cancer [J]. Cancer Lett, 2013, 335(1): 41-51.\u003c/li\u003e\n\u003cli\u003eWOLPIN B M, HEZEL A F, ABRAMS T, et al. Oral mTOR inhibitor everolimus in patients with gemcitabine-refractory metastatic pancreatic cancer [J]. J Clin Oncol, 2009, 27(2): 193-8.\u003c/li\u003e\n\u003cli\u003eQIN C, YANG G, YANG J, et al. Metabolism of pancreatic cancer: paving the way to better anticancer strategies [J]. Mol Cancer, 2020, 19(1): 50.\u003c/li\u003e\n\u003cli\u003eD\u0026apos;AMICO D, MOTTIS A, POTENZA F, et al. The RNA-Binding Protein PUM2 Impairs Mitochondrial Dynamics and Mitophagy During Aging [J]. Mol Cell, 2019, 73(4): 775-87.e10.\u003c/li\u003e\n\u003cli\u003eLEE S, KOPP F, CHANG T C, et al. Noncoding RNA NORAD Regulates Genomic Stability by Sequestering PUMILIO Proteins [J]. Cell, 2016, 164(1-2): 69-80.\u003c/li\u003e\n\u003cli\u003eMUNSCHAUER M, NGUYEN C T, SIROKMAN K, et al. The NORAD lncRNA assembles a topoisomerase complex critical for genome stability [J]. Nature, 2018, 561(7721): 132-6.\u003c/li\u003e\n\u003cli\u003eELGUINDY M M, KOPP F, GOODARZI M, et al. PUMILIO, but not RBMX, binding is required for regulation of genomic stability by noncoding RNA NORAD [J]. Elife, 2019, 8.\u003c/li\u003e\n\u003cli\u003eKOPP F, ELGUINDY M M, YALVAC M E, et al. PUMILIO hyperactivity drives premature aging of Norad-deficient mice [J]. Elife, 2019, 8.\u003c/li\u003e\n\u003cli\u003eBICKENBACH K A, VEERAPONG J, SHAO M Y, et al. Resveratrol is an effective inducer of CArG-driven TNF-alpha gene therapy [J]. Cancer Gene Ther, 2008, 15(3): 133-9.\u003c/li\u003e\n\u003cli\u003eMARIGNOL L, COFFEY M, HOLLYWOOD D, et al. Radiation to control transgene expression in tumors [J]. Cancer Biol Ther, 2007, 6(7): 1005-12.\u003c/li\u003e\n\u003cli\u003eJEONG S H, KIM H J, RYU H J, et al. ZnO nanoparticles induce TNF-\u0026alpha; expression via ROS-ERK-Egr-1 pathway in human keratinocytes [J]. J Dermatol Sci, 2013, 72(3): 263-73.\u003c/li\u003e\n\u003cli\u003eVAISH V, PIPLANI H, RANA C, et al. NSAIDs may regulate EGR-1-mediated induction of reactive oxygen species and non-steroidal anti-inflammatory drug-induced gene (NAG)-1 to initiate intrinsic pathway of apoptosis for the chemoprevention of colorectal cancer [J]. Mol Cell Biochem, 2013, 378(1-2): 47-64.\u003c/li\u003e\n\u003cli\u003eKNUDSEN A M, EILERTSEN I, KIELLAND S, et al. Expression and prognostic value of the transcription factors EGR1 and EGR3 in gliomas [J]. Sci Rep, 2020, 10(1): 9285.\u003c/li\u003e\n\u003cli\u003eFENG Y H, SU Y C, LIN S F, et al. Oct4 upregulates osteopontin via Egr1 and is associated with poor outcome in human lung cancer [J]. BMC Cancer, 2019, 19(1): 791.\u003c/li\u003e\n\u003cli\u003ePARK S Y, KIM J Y, LEE S M, et al. Expression of early growth response gene-1 in precancerous lesions of gastric cancer [J]. Oncol Lett, 2016, 12(4): 2710-5.\u003c/li\u003e\n\u003cli\u003eLIU J, GROGAN L, NAU M M, et al. Physical interaction between p53 and primary response gene Egr-1 [J]. Int J Oncol, 2001, 18(4): 863-70.\u003c/li\u003e\n\u003cli\u003eXIAO D, CHINNAPPAN D, PESTELL R, et al. Bombesin regulates cyclin D1 expression through the early growth response protein Egr-1 in prostate cancer cells [J]. Cancer Res, 2005, 65(21): 9934-42.\u003c/li\u003e\n\u003cli\u003eKUO P L, CHEN Y H, CHEN T C, et al. CXCL5/ENA78 increased cell migration and epithelial-to-mesenchymal transition of hormone-independent prostate cancer by early growth response-1/snail signaling pathway [J]. J Cell Physiol, 2011, 226(5): 1224-31.\u003c/li\u003e\n\u003cli\u003eCHEN H A, KUO T C, TSENG C F, et al. Angiopoietin-like protein 1 antagonizes MET receptor activity to repress sorafenib resistance and cancer stemness in hepatocellular carcinoma [J]. Hepatology, 2016, 64(5): 1637-51.\u003c/li\u003e\n\u003cli\u003eDEL POGGETTO E, HO I L, BALESTRIERI C, et al. Epithelial memory of inflammation limits tissue damage while promoting pancreatic tumorigenesis [J]. Science, 2021, 373(6561): eabj0486.\u003c/li\u003e\n\u003cli\u003eKLEMM S L, SHIPONY Z, GREENLEAF W J. Chromatin accessibility and the regulatory epigenome [J]. Nat Rev Genet, 2019, 20(4): 207-20.\u003c/li\u003e\n\u003cli\u003eBUENROSTRO J D, GIRESI P G, ZABA L C, et al. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position [J]. Nat Methods, 2013, 10(12): 1213-8.\u003c/li\u003e\n\u003cli\u003eCORCES M R, GRANJA J M, SHAMS S, et al. The chromatin accessibility landscape of primary human cancers [J]. Science, 2018, 362(6413).\u003c/li\u003e\n\u003cli\u003eALONSO-CURBELO D, HO Y J, BURDZIAK C, et al. A gene-environment-induced epigenetic program initiates tumorigenesis [J]. Nature, 2021, 590(7847): 642-8.\u003c/li\u003e\n\u003cli\u003eVERMA P, ZHOU Y, CAO Z, et al. ALC1 links chromatin accessibility to PARP inhibitor response in homologous recombination-deficient cells [J]. Nat Cell Biol, 2021, 23(2): 160-71.\u003c/li\u003e\n\u003cli\u003eSAQCENA M, LEANDRO-GARCIA L J, MAAG J L V, et al. SWI/SNF Complex Mutations Promote Thyroid Tumor Progression and Insensitivity to Redifferentiation Therapies [J]. Cancer Discov, 2021, 11(5): 1158-75.\u003c/li\u003e\n\u003cli\u003eARRUABARRENA-ARISTORENA A, MAAG J L V, KITTANE S, et al. FOXA1 Mutations Reveal Distinct Chromatin Profiles and Influence Therapeutic Response in Breast Cancer [J]. Cancer Cell, 2020, 38(4): 534-50.e9.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Correlation between PUM2 expression and clinicopathological parameters in pancreatic cancer\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"463\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 116px;\"\u003e\n \u003cp\u003eVariables\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 272px;\"\u003e\n \u003cp\u003ePUM2 expression\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003ep value\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003en=44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003eLow expression\u003cbr\u003e\u0026nbsp;（n=16）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eHigh expression\u003cbr\u003e\u0026nbsp;（n=28）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 463px;\"\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 463px;\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003e＜60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026ge;60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 463px;\"\u003e\n \u003cp\u003eLocation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003eHead\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003eBody/Tail\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 463px;\"\u003e\n \u003cp\u003eNerve invasion\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 463px;\"\u003e\n \u003cp\u003eDifferential dreege\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003eG1-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.031\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003eG3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 463px;\"\u003e\n \u003cp\u003eT stage\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003eT1-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.029\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003eT3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 463px;\"\u003e\n \u003cp\u003eN stage\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003eN0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.113\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003eN1-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 463px;\"\u003e\n \u003cp\u003eTNM stage\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003eI/IIA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.113\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003eIIB/III\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eTable 2. Univariate and multivariate Cox regression analysis of prognostic risk factors in patients with pancreatic cancer\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"652\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 113px;\"\u003e\n \u003cp\u003eVariabes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 47px;\"\u003e\n \u003cp\u003en\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 246px;\"\u003e\n \u003cp\u003eUnivariate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 246px;\"\u003e\n \u003cp\u003eMultivariate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003eHR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e95%CI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003ep value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eHR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e95%CI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003ep value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 652px;\"\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.238\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e0.494-3.102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.649\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 652px;\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026ge;60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.562\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e0.250-1.2611\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.162\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e＜60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 652px;\"\u003e\n \u003cp\u003eLocation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eBody/Tail\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.409\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e0.174-0.964\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cspan style=\"color: rgb(226, 80, 65);\"\u003e0.041\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e2.140\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e0.281-1.394\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.252\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eHead\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 652px;\"\u003e\n \u003cp\u003eNerve invasion\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e2.248\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e0.992-5.097\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.052\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 652px;\"\u003e\n \u003cp\u003eDifferential degree\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eG3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.984\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e0.853-4.615\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.112\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eG1-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 652px;\"\u003e\n \u003cp\u003eT stage\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eT3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e2.253\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e0.949-5.353\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.066\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eT1-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 652px;\"\u003e\n \u003cp\u003eN stage\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eN1-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e2.797\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e1.263-6.197\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cspan style=\"color: rgb(226, 80, 65);\"\u003e0.011\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e2.140\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e0.959-4.777\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.063\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eN0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 652px;\"\u003e\n \u003cp\u003eTNM stage\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eIIB/III\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e2.797\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e1.263-6.197\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eI/IIA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 652px;\"\u003e\n \u003cp\u003ePUM2 expression\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eHigh expression\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e16.671\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e4.882-56.922\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cspan style=\"color: rgb(226, 80, 65);\"\u003e＜0.001\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e15.280\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e4.461-52.336\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cspan style=\"color: rgb(226, 80, 65);\"\u003e＜0.001\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eLow expression\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Pancreatic cancer, RNA binding protein, Chemoresistance, Gemcitabine, PUM2, ITGA3, EGR1","lastPublishedDoi":"10.21203/rs.3.rs-5312328/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5312328/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePancreatic cancer (PC) has insidious onset, high malignancy and poor prognosis. Gemcitabine (GEM) is one of the first-line chemotherapy drugs for PC. However, resistance for GEM has always been a bottleneck problem leading to recurrence and death of PC patients. RNA-binding proteins (RBPs) are a kind of important proteins that regulate transportation, splicing, stability and translation of RNA. Abnormal expression of RBP often leads to a series of abnormal accumulation or degradation of downstream RNA resulting in various diseases. However, there is a lack of systematic study on whether RBPs play roles in GEM resistance of PC. Therefore, it is of great significance to explore RBPs and their specific molecular mechanisms that play an important role in GEM resistance of PC for further understanding and solving GEM resistance of PC.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eRBPs closely related to GEM resistance of PC were screened based on transcriptome sequencing, siRNA library proliferation and GEM resistance test results. Relationship between expression level of PUM2 and clinicopathological variables was evaluated by immunohistochemical (IHC) staining of PC tissue chip. SRB proliferation assay, GEM drug resistance assay and transwell cell migration assay were used to detect the effects of PUM2 on the malignant biological behaviors of PC cells \u003cem\u003ein vitro\u003c/em\u003e. Mice subcutaneous xenograft model was used to explore the effect of PUM2 \u003cem\u003ein vivo\u003c/em\u003e. Furthermore, RIP-seq and RNA-seq were combined to explore the downstream mRNAs regulated by PUM2 in PC cells, and the regulation effect of PUM2 on downstream mRNAs was verified by qRT-PCR, Western Blot, RIP-qPCR, actinomycin D RNA stability assay, dual luciferase gene reporter assay and rescue experiments. Finally, transcription factors with mutual regulation relationship with PUM2 were screened by integrating data of RIP-seq, RNA-seq and JSAPAR database, and the regulatory relationship between the transcription factor EGR1 and PUM2 was verified by qRT-PCR, Western Blot, RIP-qPCR and rescue experiments.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSeveral RBPs were found highly expressed in GEM resistant PC cell line. We screened out RNA-binding protein PUM2 as the most related RBP with GEM resistance of PC by siRNA library. IHC of PC tissue chip suggested that high expression of PUM2 was an independent risk factor for poor prognosis of PC patients. \u003cem\u003eIn vitro\u003c/em\u003e function experiments showed that PUM2 could promote proliferation, migration and resistance to GEM of PC cells. \u003cem\u003eIn vivo\u003c/em\u003e experiments showed that knockdown of PUM2 inhibited the growth of subcutaneous transplanted tumor in mice and increased sensitivity to GEM. Further, RNA-seq and RIP-seq were combined to explore the regulation role of PUM2 on downstream RNAs that promoted GEM resistance in PC. We found that PUM2 up-regulated mRNA stability of key genes (ITGA3, ADAM17, ASAP1, etc.) in the focal adhesion pathway. ITGA3 was verified to be the most significant downstream mRNA of PUM2 regulating GEM resistance in PC by rescue experiments \u003cem\u003ein vitro\u003c/em\u003e, and PUM2 could stabilize ITGA3 mRNA by binding to PUM binding element (PBE) in the 3'UTR region of ITGA3 mRNA. Finally, we found the mutual regulation relationship between transcription factor EGR1 and PUM2, that is PUM2 binding to 3'UTR region of EGR1 mRNA, and EGR1 binding to promoter region of PUM2 gene, resulting in a cascade effect amplifying the role of PUM2 in PC chemoresistance.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eRNA-binding protein PUM2 is closely related to the prognosis of PC patients. PUM2 promoted GEM resistance of PC by regulating mRNA stability of ITGA3 and other genes in focal adhesion pathway, and there was positive feedback regulation between PUM2 and transcription factor EGR1. The discovery of EGR1/PUM2/ITGA3 axis provided a solid experimental basis for the selection of chemotherapy regiments for PC patients and exploration of combined regimens to reverse GEM resistance in the future.\u003c/p\u003e","manuscriptTitle":"RNA binding protein Pumilio2 promotes chemoresistance of pancreatic cancer via focal adhesion pathway and interacting with transcription factor EGR1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-20 12:07:43","doi":"10.21203/rs.3.rs-5312328/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2024-11-11T05:02:44+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-11-01T00:30:42+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-31T13:54:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-28T10:16:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2024-10-22T09:46:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8e83643e-6859-421c-98c9-acfdf8cb5caa","owner":[],"postedDate":"November 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-24T16:25:14+00:00","versionOfRecord":{"articleIdentity":"rs-5312328","link":"https://doi.org/10.1007/s00018-025-05599-8","journal":{"identity":"cellular-and-molecular-life-sciences","isVorOnly":false,"title":"Cellular and Molecular Life Sciences"},"publishedOn":"2025-02-17 15:57:42","publishedOnDateReadable":"February 17th, 2025"},"versionCreatedAt":"2024-11-20 12:07:43","video":"","vorDoi":"10.1007/s00018-025-05599-8","vorDoiUrl":"https://doi.org/10.1007/s00018-025-05599-8","workflowStages":[]},"version":"v1","identity":"rs-5312328","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5312328","identity":"rs-5312328","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}