Genome-scale activation screen reveals lncRNA HNF1A-AS1 as a novel therapeutic target for pancreatic cancer metastasis

Genome-scale activation screen reveals lncRNA HNF1A-AS1 as a novel therapeutic target for pancreatic cancer metastasis | 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 Article Genome-scale activation screen reveals lncRNA HNF1A-AS1 as a novel therapeutic target for pancreatic cancer metastasis Shan Lei, Zhixue Zhang, Zhirui Zeng, Wenpeng Cao, Yating Sun, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5388928/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Long non-coding RNAs (LncRNAs) have emerged as pivotal biomarkers and regulators across various cancers. In pancreatic cancer (PC), however, the mechanisms underlying the expression and functional roles of lncRNAs remain inadequately elucidated. Methods CRISPR/CRISPR-associated protein 9 (Cas9) single-guide RNA (sgRNA)-pooled lncRNA libraries were used to screen for the critical lncRNAs regulating PC metastasis. The expression levels of lncRNA HNF1A-AS1 were quantified in PC cell lines and clinical samples using qRT-PCR. Investigations into HNF1A-AS1's impact on PC cell migration and invasion were conducted through both loss-of-function and gain-of-function approaches. A range of techniques, including fluorescence in situ hybridization (FISH), mRNA sequencing, RNA immunoprecipitation (RIP), bioinformatics analysis, dual-luciferase reporter assays, RNA pull-down assays, ChIP-PCR, and rescue experiments, were employed to unravel the competitive endogenous RNA (ceRNA) network regulated by HNF1A-AS1. Results The research identified HNF1A-AS1 as a novel and influential lncRNA that acts as a pro-metastatic factor in PC. Compared to normal controls, HNF1A-AS1 levels were significantly elevated in PC cell lines and tissue samples. Elevated HNF1A-AS1 expression correlated with increased lymph node metastasis and poorer overall survival in patients with PC. Knocking down HNF1A-AS1 substantially reduced metastasis, whereas its overexpression exacerbated it. Mechanistically, HNF1A-AS1 promotes an oncogenic splice switch from the standard isoform CD44s to the variant isoform CD44v (3–10), acting as a scaffold for the binding of CD44 pre-mRNA to U2SURP. The levels of HNF1A-AS1 and CD44v (3–10) serve as indicators of poor prognosis. Furthermore, SNAI2 was shown to specifically bind to the HNF1A-AS1 promoter, thereby activating its transcription. Antisense oligonucleotides (ASOs) targeting HNF1A-AS1 also significantly inhibited cancer metastasis. Conclusions SNAI2’s role in enhancing HNF1A-AS1 transcription underscores the critical function of HNF1A-AS1 in promoting PC metastasis through modulation of CD44 alternative splicing via U2SURP. Targeted silencing of HNF1A-AS1 presents a promising therapeutic avenue for patients with PC. Biological sciences/Cell biology/Cell migration/Cell invasion Biological sciences/Molecular biology/Non-coding RNAs/Long non-coding RNAs Biological sciences/Cancer/Cancer therapy/Drug development pancreatic cancer HNF1A-AS1 alternative splicing U2SURP CD44 antisense oligonucleotides Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Pancreatic cancer (PC) ranks as the fourth leading cause of cancer-related mortality globally. Despite advances in treatment, the 5-year survival rate for patients with PC remains below 5% [ 1 ]. The poor prognosis is largely due to the early invasion and rapid proliferation of cancer cells [ 2 , 3 ]. Thus, it is crucial to understand the molecular mechanisms underlying PC development and to develop effective therapeutic strategies. Long non-coding RNAs (LncRNAs), which exceed 200 nucleotides in length but lack coding potential, have been implicated in various biological processes, including the cell cycle, apoptosis, aging, metastasis, and chemotherapy resistance [ 4 , 5 , 6 ]. Recent research highlights the pivotal role of alternative splicing (AS) in tumorigenesis [ 7 , 8 ]. AS generates multiple RNA transcripts from a single precursor mRNA, offering a promising area for enhancing clinical diagnosis, disease prediction, and therapeutic development [ 9 , 10 ]. Dysregulation of AS can lead to diseases and impact gene stability, with significant implications for tumor biology [ 11 , 12 ]. Therefore, identifying and understanding AS-related genes in PC may provide valuable insights for effective treatment. Given the noncoding nature and diverse functionalities of long noncoding RNAs (lncRNAs), a systematic methodology is imperative to elucidate their roles in prostate cancer (PC) metastasis. Clustered regularly interspaced short palindromic repeats (CRISPR) coupled with CRISPR-associated protein 9 (Cas9) screening represents a novel technique for identifying potential essential genes or therapeutic targets through gene knockout or activation [ 13 ]. A CRISPR library comprises thousands of single-guide RNAs (sgRNAs), which can target both protein-coding genes and noncoding RNAs. Through a knockout strategy, essential genes can be discerned via negative selection [ 14 ]. Another CRISPR/Cas9 strategy developed by Zhang employs a three-component engineered protein complex capable of activating the transcription of long non-coding RNAs (lncRNAs) via single guide RNAs (sgRNAs) [ 15 ]. Utilizing a lncRNA activation library, genes that inhibit cancer metastasis can be identified through negative selection, whereas genes that drive cancer metastasis are prevalent in the positive selection results. This powerful tool facilitates the elucidation of the molecular mechanisms underlying prostate cancer (PC) metastasis and other cancer types, allowing for the consideration of previously undiscovered targets. CD44, a multifunctional transmembrane glycoprotein, is essential for maintaining normal cellular functions and has been shown to influence cancer development and progression [ 16 ]. CD44 undergoes alternative splicing to produce various isoforms with distinct functions [ 17 ]. However, the molecular mechanisms driving AS and the roles of CD44 variants in cancer remain poorly understood. This study compared CD44v (3–10) and CD44s interactomes using mass spectrometry (MS)-based proteomics. It investigated the biological role and clinical relevance of HNF1A-AS1 in cancer metastasis, examining its potential regulation of CD44 alternative splicing through U2SURP. Additionally, the study explored how CD44 variants contribute to cancer metastasis and assessed an antisense oligonucleotide (ASO) targeting HNF1A-AS1 in preclinical trials for its potential to inhibit cancer metastasis. MATERIALS AND METHODS Cell culture Normal human pancreatic epithelial cells (HPDE) and PC cell lines (BxPC-3, PANC-1, MIA PaCa-2, AsPC-1, PATU8988-S, Capan-1) were procured from DSMZ (Braunschweig, Germany). The PANC-1-Luc and AsPC-1-Luc cell lines were obtained from Procell (Wuhan, China). PANC-1 and MIA PaCa-2 cells were cultured in DMEM (Dulbecco's Modified Eagle Medium; Gibco, USA) with 10% FBS (Bioind, Israel), while HPDE, AsPC-1, Capan-1, PATU8988-S, and BxPC-3 cells were maintained in RPMI-1640 (Roswell Park Memorial Institute-1640 Medium; Gibco, USA) with 10% FBS. All cell lines were incubated at 37°C with 5% CO2 and verified by short tandem repeat profiling. Plasmid construction and cell transfection plasmids Lentiviruses for HNF1A-AS1 knockdown, overexpression, and negative controls were sourced from Genechem (Shanghai, China). Transfections of plasmids, siRNAs, or ASOs were performed using Lipofectamine 3000 (Thermo Fisher Scientific) as per the manufacturer's protocol [18]. The pcDNA3.1 vector (Addgene plasmid 52961) was used to clone CD44s, CD44v (3-10), and U2SURP coding sequences. Mutants were generated using primers with mismatched nucleotides. HNF1A-AS1 ASOs were provided by GenePharma (Shanghai, China). All sequences used in this study are summarized in Supplementary Table S1. In vitro CRISPR‒Cas9 library screen CRISPR-Cas9 long non-coding RNA (lncRNA) activation screens were conducted on pooled human CRISPR 3-plasmid lncRNA Synergistic Activation Mediator (SAM) libraries (Addgene #1000000106). The library contains 95,058 single guide RNAs (sgRNAs) targeting 10,504 lncRNAs, with around ten sgRNAs aimed at the transcription start site (TSS) of each lncRNA. Using Endura electrocompetent cells, the sgRNA library was cloned according to protocol and amplified according to the manufacturer's instructions [19]. AsPC-1 cells were transduced with dCas9-VP64 and MPHv2 and subjected to a five-day selective selection regime using the purified sgRNA library and the other two components of the SAM system: dCas9-VP64-blast and MS2-P65-HSF1. After zeocin kill curve analysis, stable clones were transduced with a sgRNA library at a multiplicity of infection (MOI) less than 0.3, ensuring that each cell only contained one sgRNA. Approximately 2×10 7 cells were harvested one week later to collect input DNA, with those remaining cells selected using Matrigel covered Boyden chambers with 300 g/mL zeocin (InvivoGen, USA). A sufficient number of cells were obtained for the next invasion selection round by removing invading and adhering cells with trypsin and culturing them. Reseeding was then performed three times in new invasion chambers. In order to ensure adequate representation of sgRNA, a minimum of 7×10 7 cells were maintained at all times. To extract total genomic DNA, the Zymo Research Quick-gDNA MidiPrep Kit (Zymo Research, USA) was used. After the third round of screening, the cells were harvested and genomic DNA was extracted. In a subsequent PCR, ten pairs of barcoded primers were used to amplify the subregions targeted by the sgRNA. The PCR products were purified using Qiagen gel kits and then analyzed for sgRNAs via next-generation paired-end sequencing (Novogene). qRT-PCR and Western blotting analysis Western blotting and qRT-PCR were conducted as previously described [20], with qRT-PCR reagents supplied by Takara (Japan). Primer sequences for qRT-PCR and antibodies for Western blotting are detailed in Table S1. Tissue microarray, in situ hybridization (ISH), and immunohistochemistry (IHC) staining Immunohistochemical staining for CD44v (3-10) followed established protocols [21]. Briefly, tissue samples were deparaffinized in xylene, rehydrated through a gradient of alcohols, and subjected to high-temperature antigen retrieval. Sections were then incubated overnight at 4°C with antibodies against CD44v (3-10) and vimentin, followed by biotinylated secondary antibodies. Visualization was achieved using DAB (3,3'-Diaminobenzidine; Dako, Mississauga, ON, Canada). HNF1A-AS1 in situ hybridization (ISH) probes were obtained from Qiagen (Hilden, Germany), and ISH was performed according to the instructions provided with the ISH kitTM (Servicebio, Wuhan, China). Tissue microarrays were deparaffinized in xylene, rehydrated with ethanol, and digested with 20 µg/ml pepsin. After hybridization with digoxigenin-labeled probes and secondary antibodies, samples were stained with DAB. RNA pulldown The MegaScript TM T7 Transcription Kit (Invitrogen) was utilized to purify truncated mutations or antisense RNA by transcribing full-length or truncated variants with T7 RNA polymerase (Roche Diagnostics, Indianapolis, USA). Biotin-labeled lncRNA was captured using streptavidin magnetic beads and incubated with PC cell lysate at 4°C for 1 hour. Eluates were then analyzed by mass spectrometry or Western blotting following separation and purification. MS2-RIP-MS/qPCR pcDNA3.1 constructs including 24 × MS2-HNF1A-AS1, 24 × MS2 empty vector, 24 × MS2-antisense-HNF1A-AS1, or 24 × MS2-HNF1A-AS1 mutants (△BR1, △BR2, △BR3, △BR4, △BR5) were co-transfected with pMS2-GST. Cells were harvested 48 hours post-transfection, and RNA-binding proteins were purified using the Magna RIP RNA Binding Protein Immunoprecipitation Kit (Millipore) for qRT-PCR analysis. MS was used to identify proteins interacting with sense and antisense HNF1A-AS1. Transwell invasion assays Invasion assays were conducted as previously described [22]. Matrigel (20 µg; BD Biosciences) was evenly applied to the upper chamber membrane. Subsequently, 2 × 10 4 cells resuspended in 200 µL of serum-free medium were seeded into the upper chamber. The lower chamber was filled with 700 µL of medium containing 10% FBS. After 24 hours of incubation, cells were fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 20 minutes, washed, and observed under an inverted microscope. Wound healing assay For migration assays, PC cells from different treatment groups were seeded in six-well plates and grown to approximately 100% confluence. Cells were scraped with a 200 µL pipette tip, and serum-containing media were removed. The cultures were then treated with mitomycin-C and incubated for 24 hours. Migration was assessed by capturing images at 0 and 24 hours post-scratching. Lymph node metastasis model assay Luciferase-expressing PC cells (2 × 10 6 cells per mouse) suspended in 150 µL of PBS were inoculated subcutaneously into the left hind foot pad of 6 to 8-week-old female immunodeficient mice. Mice were randomly assigned to treatment groups (n = 5 per group). Following intraperitoneal injection of 100 µL of D-luciferin methyl salt (150 mg/kg in PBS), mice were anesthetized with 3% isoflurane gas and monitored for tumor metastasis using the IVIS 100 imaging system (Caliper Life Sciences, Waltham, MA, USA). Mice were then euthanized, and tumors and lymph nodes were excised, fixed in 4% PFA, and subjected to histological analysis. Treatments were administered in a non-blinded manner. All animal experiments were conducted following the guidelines of the Animal Ethics Committee at Guizhou Medical University. In vivo metastasis model The splenic injection assay assessed the liver metastasis capability of various PC cell groups. A total of 2 × 10 6 luciferase-expressing PC cells in 100 µL PBS were injected into the spleens of 6-week-old female BALB/C nude mice (n = 5 per group). For evaluating lung metastasis, the same PC cells were administered via tail vein injection into female BALB/C nude mice (n = 5 per group, 2 × 10 6 cells per mouse). Mice were weighed weekly and euthanized after 12 weeks. Following tail vein injection, 100 µL of D-luciferin methyl salt (150 mg/kg in PBS) was intraperitoneally administered, and metastatic spread was monitored using the IVIS 100 Imaging System (Caliper Life Sciences, Waltham, MA, USA). Lungs and livers were excised, fixed in 4% paraformaldehyde for 24-48 hours, embedded in paraffin, and subjected to Hematoxylin-Eosin (H&E) staining to identify metastatic sites. Chromatin immunoprecipitation (ChIP)-qPCR assay ChIP assays were conducted following the SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) (9003S, CST) protocol. Cells (1 × 10 6 ) were cross-linked with 1% formaldehyde at room temperature for 10 minutes. Cell pellets were then incubated in Buffer A at 4°C for 10 minutes before nuclei were isolated by centrifugation at 5000 rpm. Nuclei were subsequently digested in Buffer B with 25 units of micrococcal nuclease (M Nase; 10011, CST) for 20 minutes at 37°C. DNA was fragmented using pulsed ultrasonication, and chromatin was collected by centrifugation at 12,000 rpm for 10 minutes. Chromatin DNA was quantified, and an equal amount was incubated overnight at 4°C with either the target antibody or a negative control IgG. Immunoprecipitated complexes were then incubated with Protein A/G magnetic beads for 2 hours at 4°C, extensively washed, and DNA was purified using spin columns. Quantitative PCR with TB Green Premix Ex Taq™ on a CFX Connect Real-Time System was used to quantify the purified DNA. Quantification was based on input and IgG-enriched signals, with values normalized to a baseline of 1. RT–PCR detection For reverse-transcription PCR (RT-PCR) assays, primers were designed using Primer Premier 5 based on alternative splicing event (ASE) analysis to detect exon skipping and validate RNA splicing. The RT-PCR protocol consisted of an initial denaturation at 95°C for 2 minutes, followed by 36 cycles of denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, and elongation at 72°C for 1 minute. A final elongation step was performed at 72°C for 5 minutes, with the reaction held at 4°C. PCR products were separated on 2% agarose gels stained with 0.1‰ ethidium bromide and visualized using a GleUV system (Baygene Biotech). Band intensities were quantified with ImageJ. Statistical analysis Each in vitro experiment was replicated three times. Statistical analyses were conducted using GraphPad Prism 8.0 (GraphPad Software Inc., La Jolla, CA, USA) and Student's t-test. Survival analysis was performed with the Kaplan-Meier method and log-rank test using Statistical Package for the Social Sciences (SPSS) software (SPSS Inc., Chicago, IL, USA). Data are presented as mean ± S.D. from three independent experiments, with statistical significance determined at a P-value of < 0.05. RESULT Expression of HNF1A-AS1 and its relationship to clinical parameters in patients with PC Key lncRNAs involved in cancer metastasis were identified through RNA sequencing of three metastatic tumor tissues and three primary tumor tissues (Figure S1A). Of the 287 differentially expressed lncRNAs (log2 FC > 1), 146 were downregulated and 140 upregulated in metastatic tissues (Figure S1B, Table S2). We implemented a comprehensive screening strategy utilizing a CRISPR/Cas9 lncRNA SAM pooled library, which comprised 96,458 sgRNAs targeting the transcription start sites (TSSs) of 10,504 distinct lncRNAs [15]. The human pancreatic cancer cell line AsPC-1 served as the in vitro model for CRISPR activation (CRISPRa) screening to identify potential lncRNA regulators. The cell population was bifurcated into two groups: one designated as the control (Input) and the other subjected to three rounds of invasion selection (Cas9) using Matrigel-coated Boyden chambers. Genomic DNA was extracted from both cell populations, and next-generation sequencing was employed to quantify the read counts of each sgRNA (Figure 1A). The Cas9 cells demonstrated an increased invasive capacity relative to the input cells (Figure S1C). Subsequent next-generation sequencing of genomic DNA indicated a substantial reduction in the diversity of sgRNAs within the Cas9 cells, identifying 31 significantly depleted sgRNAs that target 45 genes. Moreover, an integrated analysis combining the 76 genes identified from the CRISPR/Cas9 screening with the 287 differentially expressed lncRNAs in metastatic tumor tissues yielded a shortlist of 10 overlapping lncRNAs (Figure 1B). Analysis of 80 human PC tissues and adjacent noncancerous samples via qRT-PCR detected the expression levels of the 10 lncRNAs, with HNF1A-AS1 showing markedly higher expression in PC tissues compared to adjacent noncancerous tissues, thereby becoming the focal point of this study (Figure 1C, Figure S1D). In 50 of 77 (65%) clinical sample pairs, HNF1A-AS1 expression was significantly elevated in tumor tissues relative to matched adjacent non-tumor tissues (Figure 1D). The correlation between HNF1A-AS1 expression and clinicopathological parameters of PC was further explored, revealing a significant positive association with perineural invasion (Figure 1E), distant metastasis (Figure 1F), and lymph node metastasis (Figure 1G). Other factors, such as gender, age, differentiation grade, tumor site, and tumor size, did not show significant differences (Table S3). In situ hybridization staining confirmed that HNF1A-AS1 levels were substantially higher in PC tumor tissues than in adjacent non-tumor tissues (Figure 1H). Patients were categorized into high and low HNF1A-AS1 expression groups based on median expression levels. Kaplan-Meier survival analysis, supplemented by the log-rank test, indicated that patients with lower HNF1A-AS1 expression exhibited improved overall survival compared to those with higher levels (Figure 1I). ROC curve analysis of 80 paired tissue samples yielded an area under the curve (AUC) of 0.8786, suggesting HNF1A-AS1's potential as a diagnostic marker for PC (Figure 1J). Additionally, qRT-PCR results demonstrated significantly higher HNF1A-AS1 expression in PC cell lines with high metastatic potential (AsPC-1, Capan-1, PATU8988-S) compared to those with lower metastatic capability (BxPC-3, PANC-1, MIA PaCa-2) and human pancreatic epithelial immortalized cells (HPDE) (Figure 1K). Upregulation of HNF1A-AS1 in PC promotes cancer metastasis To elucidate the biological role of HNF1A-AS1, low-metastasis PC cell lines (BxPC-3, PANC-1) with HNF1A-AS1 overexpression and high-metastasis PC cell lines (AsPC-1, Capan-1) with HNF1A-AS1 knockdown were developed (Figure 2S). Wound-healing assays demonstrated that HNF1A-AS1 overexpression enhanced PC cell migration, while HNF1A-AS1 knockdown significantly impeded migration (Figure 2A). Transwell invasion assays confirmed that overexpression of HNF1A-AS1 promoted invasion, whereas its knockdown reduced invasive capabilities (Figure 2B). Analysis of EMT marker expression revealed decreased E-cadherin levels and increased vimentin and N-cadherin levels, which facilitated cell migration and invasion (Figure 2C). Additionally, popliteal lymph node metastasis models indicated that HNF1A-AS1 overexpression resulted in more pronounced lymphadenopathy compared to controls, while knockdown led to reduced lymphadenopathy (Figure 2D). In vivo bioluminescence imaging showed stronger signals in nude mice intravenously injected with HNF1A-AS1 overexpressing PC cells, in contrast to those with knockdown (Figure 2E). Similarly, liver bioluminescence signals were more intense in mice subjected to splenic injection of HNF1A-AS1 overexpressing PC cells, whereas knockdown resulted in diminished signals (Figure 2F). HNF1A-AS1 interacts with U2SURP to exert its biological function The subcellular localization of HNF1A-AS1 was examined through nuclear-cytoplasmic fractionation and FISH assays, revealing HNF1A-AS1 is mainly localized in the nucleus (Figure S3A-B). To elucidate the molecular mechanisms underlying HNF1A-AS1's role in cancer metastasis, RNA pulldown assays coupled with MS analysis were conducted to identify its binding proteins (Figure 3A, Table S4). KEGG pathway analysis highlighted the spliceosome signaling pathway as significantly enriched among the associated proteins (Figure 3B). Integration with the splicing factor family identified CDC5L, SF3B2, HNRNPM, U2SURP, and PCBP1 as key candidates for further investigation (Figure 3C). RNA pull-down assays confirmed the binding of HNF1A-AS1 to these proteins, with U2SURP showing the most significant interaction in Capan-1 and AsPC-1 cells (Figure 3D, Figure S3C). RIP assays further validated the enrichment of HNF1A-AS1 in U2SURP immunoprecipitates (Figure 3E), and co-localization of HNF1A-AS1 with U2SURP was observed in PC cells (Figure 3F). To pinpoint the regions of HNF1A-AS1 responsible for binding U2SURP, a series of deletion mutants were constructed: △BR1 (1-500 nt), △BR2 (501-1000 nt), △BR3 (1001-1500 nt), △BR4 (1501-2000 nt), and △BR5 (2001-2455 nt) (Figure 3G). RNA pulldown assays demonstrated that binding to U2SURP is confined to the 1001-1500 nt region (Figure 3H). Further analysis using truncated binding motif mutants, including △RRM and △CID, revealed that deletion of the RRM domain disrupted the HNF1A-AS1-U2SURP interaction (Figure 3I-J), underscoring the critical role of the RRM region in this binding association. Subsequently, investigations into U2SURP's role in PC metastasis were initiated. HNF1A-AS1 acts as a scaffold to promote the binding of U2SURP to CD44 pre-mRNA Functional assays demonstrated that U2SURP-deficient impairs the ability of invasion and metastasis of pancreatic cancer cells overexpressing HNF1A-AS1 (Figure S4A-B), underscoring U2SURP's critical role in facilitating cancer metastasis through HNF1A-AS1. Subsequent investigations into the HNF1A-AS1-U2SURP interaction revealed that modifying HNF1A-AS1 did not impact U2SURP's expression levels (Figure S4C). Given U2SURP's role as a splicing factor, it was hypothesized that HNF1A-AS1 might modulate U2SURP-mediated alternative mRNA splicing. To assess HNF1A-AS1's involvement in AS, deep RNA-seq was performed on HNF1A-AS1 knockdown AsPC-1 cells, followed by rMATS analysis of splicing events (Figure 4A). The analysis revealed significant changes in AS profiles post-HNF1A-AS1 knockdown, identifying 1593 ASEs across 1451 genes (Table S5). The majority of ASEs were skipped exons (61.9%), followed by alternative 3' splice sites (14%), mutually exclusive exons (12.68%), alternative 5' splice sites (8.1%), and retained introns (3.4%) (Figure 4B). Notably, TIMMDC1, RER1, PSMA4, and HROB emerged as top candidates for alternative splicing, suggesting that HNF1A-AS1 may act as a scaffold to influence U2SURP binding to these targets. PCR primer pairs were designed to target adjacent constitutive exons in genes with skipped exons regulated by HNF1A-AS1. HNF1A-AS1 knockdown in AsPC-1 and Capan-1 cells resulted in increased in exon 7 skipping of CD44 (Figure 4D, Figure S4D), indicating that HNF1A-AS1 might modulate splicing by acting as a scaffold for U2SURP. MS2-RIP-qPCR assays confirmed that HNF1A-AS1 interacts with CD44 pre-mRNA but not with TIMMDC1, RER1, PSMA4, or HROB (Figure 4E, Figure S5), suggesting CD44 as a potential downstream target of HNF1A-AS1. RIP-qPCR assays further demonstrated that U2SURP binds to CD44 pre-mRNA, with the RRM domain of U2SURP essential for this interaction (Figure 4F). MS2-RIP-qPCR experiments, employing full-length and truncated HNF1A-AS1 fragments (△BR1, △BR2, △BR3, △BR4, △BR5), pinpointed the BR3 region as crucial for HNF1A-AS1's binding to CD44 pre-mRNA (Figure 4G). Additionally, gain- and loss-of-function experiments, combined with RNA immunoprecipitation using an anti-U2SURP antibody, demonstrated that HNF1A-AS1 significantly enhances U2SURP's binding to CD44 pre-mRNA (Figure 4H). These findings illustrate that HNF1A-AS1 facilitates the interaction between U2SURP and CD44 pre-mRNA, thereby modulating splicing events. U2SURP-induced alternative splicing of CD44 mediates the role of HNF1A-AS1 in facilitating cancer metastasis The CD44 gene undergoes extensive alternative splicing, generating various isoforms, including the standard isoform CD44s and multiple variant isoforms, CD44v (3-10) (Figure 5A). Gain- and loss-of-function experiments revealed that HNF1A-AS1 upregulates the expression level of CD44v (3-10) while downregulating CD44s (Figure 5B, C). In addition, U2SURP knockdown in PC cells led to the upregulation of CD44s and the downregulation of CD44v (3-10) (Figure 5D). Analysis of 80 PC tumor tissues alongside matched adjacent normal tissues indicated that CD44s expression was lower and CD44v (3-10) expression was higher in tumors, suggesting that HNF1A-AS1 and U2SURP orchestrate CD44 splicing to favor the production of CD44v (3-10) isoform (Figure 5E). Immunohistochemistry of 31 tumor and adjacent non-tumor tissues further demonstrated a marked increase in CD44v (3-10) expression in PC tissues compared to adjacent normal tissues (Figure 5F). Elevated CD44v (3-10) levels correlated with perineural invasion, distant metastasis, and lymph node metastasis (Table S5). Both TCGA and our dataset revealed that patients with lower CD44v (3-10) expression exhibited longer survival compared to those with higher levels (Figure 5G-H). These findings suggest that HNF1A-AS1-mediated alternative splicing of CD44, shifting from CD44s to CD44v (3-10), may serve as a marker for poor prognosis in patients with PC. To investigate the functional role of CD44v (3-10), cell lines ectopically expressing CD44s (3-10) or CD44v (3-10) were generated (Figure S6A). Invasion and wound healing assays demonstrated that CD44v (3-10) significantly enhanced the ability of PC cell invasion and metastasis compared to CD44s (Figure S6B-C). Notably, CD44v (3-10) overexpression effectively counteracted the suppression of PC invasion and metastasis induced by HNF1A-AS1 knockdown, both in vivo and in vitro (Figure I-K). These results underscore the role of HNF1A-AS1 in interacting with U2SURP to modulate CD44 splicing, thereby promoting cancer metastasis. HNF1A-AS1 was a downstream gene of SNAI2 To further elucidate the regulatory networks involving HNF1A-AS1, the interaction between HNF1A-AS1 and SNAI2 was examined. Motif sequences for SNAI2 were sourced from the JASPAR database (Figure 6A), revealing five predicted binding sites on the HNF1A-AS1 promoter (Figure 6B). PCR primers, designed to span approximately 200 bp of these binding and transcription sites, facilitated subsequent analysis (Figure 6C). ChIP-qPCR demonstrated that Flag-tagged SNAI2 successfully precipitated and amplified the HNF1A-AS1 promoter sequence, confirming the direct binding of SNAI2 to this region (Figure 6D). To investigate SNAI2's effect on transcriptional activity, promoter sequences, including wild-type (Wt) and various mutant binding sites (Mut), were cloned into the pGL4.20 plasmid and transfected into both control and SNAI2-overexpressing PC cells (Figure 6E). Luciferase assays revealed that SNAI2 overexpression enhanced the HNF1A-AS1 promoter activity without altering the SNAI2 binding sites (Figure 6F). This suggests that SNAI2 directly binds to the HNF1A-AS1 promoter to drive its transcription. RT-qPCR further confirmed that SNAI2 overexpression in PC cells elevated HNF1A-AS1 levels, while SNAI2 knockdown reduced its expression (Figure 6G). FISH and immunofluorescence staining of tissue samples also demonstrated co-expression of SNAI2, HNF1A-AS1, and U2SURP in PC tissues (Figure 6H). Specific ASOs suppress tumor progression by controlling HNF1A-AS1 circularization and secretion The second-generation ASOs designed target HNF1A-AS1, utilizing phosphorothioate backbones and modified oligoribonucleotides to lower intracellular levels of HNF1A-AS1 (Figure 7A). The efficacy of ASO1-HNF1A-AS1 was evaluated both in vitro and in vivo . Transwell invasion and wound healing assays demonstrated that ASO1-HNF1A-AS1 markedly inhibited the invasion and migration of PC cells (Figure 7B-C). Western blot analysis confirmed a significant reduction in epithelial-mesenchymal transition (EMT) due to ASO1-HNF1A-AS1 treatment (Figure 7D). Further examination of tumor metastasis in a xenograft model revealed that ASO1-HNF1A-AS1 effectively diminished liver metastasis when PC cells were injected into the mouse spleen, as monitored by IVIS imaging (Figure 7F). Similarly, ASO1-HNF1A-AS1 treatment substantially reduced lung metastases in an experimental lung metastasis model (Figure 7G) and significantly inhibited lymph node metastasis in the footpad inoculation model (Figure 7H). No notable changes in organ morphology or levels of key blood parameters—such as monocytes, white blood cells, lymphocytes, granulocytes, red blood cells, hemoglobin, platelets, hematocrit, aspartate aminotransferase, and alanine aminotransferase—were observed in ASO1-HNF1A-AS1-treated mice, indicating the absence of toxic effects (Figure S7). Collectively, these results suggest that ASO1-HNF1A-AS1 holds promise as a novel therapeutic approach for cancer treatment. DISCUSSION In recent years, lncRNAs have emerged as pivotal players in various aspects of cancer biology, including tumor metabolism, proliferation, metastasis, and chemotherapy resistance, garnering significant attention in cancer research [ 23 , 24 ]. To evaluate the role of HNF1A-AS1 in PC, its expression levels were measured using qRT-PCR and in situ hybridization (ISH) across bile duct adjacent/primary tissues, normal/cancer cell lines, and metastatic tumor samples. Prognostic implications were further explored through survival analysis. Both in vivo and in vitro studies revealed that HNF1A-AS1 knockdown substantially impaired cell migration and invasion, suggesting that HNF1A-AS1 may function as an oncogene in PC and is critical to disease progression. This study also investigated the mechanism underlying the upregulation of HNF1A-AS1 in PC cells. It was found that SNAI2 specifically binds to the promoter region of HNF1A-AS1, elucidating how SNAI2 contributes to its expression. Proteomic analyses, including RNA-seq, RNA pull-down assays, and mass spectrometry, identified U2 snRNP-associated SURP motif-containing protein (U2SURP) as a key interactor with HNF1A-AS1. U2SURP, a serine/arginine-rich protein and a component of the 17S U2 snRNP complex, features a highly phosphorylated SR domain at the carboxy terminus and an RNA recognition motif (RRM) at the amino terminus [ 25 , 26 ]. Despite its importance, the regulatory mechanisms and biological functions of U2SURP remain underexplored. Previous research by De Maio et al. has shown that U2SURP regulates splicing and gene expression by interacting with other splicing factors, such as RBM17 and CHEP [ 27 ]. AS is a vital process that enhances gene expression and transcriptome diversity in eukaryotic cells, enabling a single precursor mRNA to generate multiple mRNA and protein variants [ 28 , 29 ]. Over 95% of human gene transcripts with multiple exons undergo AS [ 30 ], with different splice variants being expressed across various tissues and cells [ 31 ]. Dysregulation of AS can contribute to a range of human diseases. This study demonstrates that the lncRNA HNF1A-AS1 and U2SURP are involved in CD44 alternative splicing, leading to EMT activation. The spliceosomal variants of CD44 exhibit different roles across various tumors [ 17 , 30 ]. Some studies suggest that the transition from CD44v to CD44s can facilitate tumor progression [ 32 , 33 ], while others indicate that CD44v promotes tumor development [ 34 , 35 ]. Conversely, the conversion from CD44s to CD44v has been associated with accelerated tumor progression [ 36 , 37 ]. This variability in function highlights the controversy surrounding the roles of different CD44 splice isoforms in PC [ 38 ]. Our research identified CD44v (3–10) as a key player in tumor progression. Knockdown of HNF1A-AS1 significantly reduced CD44 precursor mRNA splicing to CD44v (3–10), thereby inhibiting tumor advancement. Overexpression of CD44v (3–10) in PC cells further confirmed its role in promoting tumor progression, suggesting that targeting the alternative splicing of CD44v (3–10) could offer a novel therapeutic strategy. Metastatic cancer accounts for approximately 90% of cancer-related deaths worldwide, yet the mechanisms underlying cancer metastasis remain poorly understood [ 39 , 40 ]. Current treatments for metastatic cancer are inadequate. Our study reveals that HNF1A-AS1 enhances the alternative splicing of CD44 pre-mRNA by interacting with the splicing factor U2SURP, leading to increased production of CD44v (3–10). This splice variant, a novel regulator of cancer metastasis, promotes tumor invasion and dissemination, positioning it as a potential therapeutic target. Two decades ago, the ability of ASOs to influence RNA processing and protein expression was first discovered. Recent clinical trials have demonstrated the effectiveness of ASO-based therapies [ 41 , 42 , 43 ]. Our study shows that ASOs targeting HNF1A-AS1 significantly inhibit tumor invasion and metastasis in both cellular and animal models. In conclusion, this study provides the first evidence that SNAI2 specifically binds to the HNF1A-AS1 promoter and enhances its expression in PC. The interaction between HNF1A-AS1 and U2SURP facilitates the alternative splicing of CD44, shifting from the standard isoform CD44s to the variant CD44v (3–10), which promotes cancer invasion and metastasis. This underscores the critical role of CD44 splicing in cancer progression and suggests that ASOs targeting HNF1A-AS1 could be a promising approach for treating PC. Declarations Acknowledgments Not applicable. Conflict of Interest The authors declare that there are no potential conflicting interests associated with their research. Author Contribution Tengxiang Chen designed the experiments and drafted the manuscript; Shan Lei, Zhirui Zeng, Zhixue Zhang, Yating Sun, Wenpeng Cao, Jigang Pan, Tuo Zhang, Yingmin Wu and Dahuan Li conducted and processed the data. All authors reviewed the final version of the manuscript and granted approval. Ethics Statement The Human Research Ethics Review Committee of Guizhou Medical University approved the application of these clinical samples, which was performed according to the tenets of the Declaration of Helsinki. The animal experiments were approved by Animal Ethics Committee of Guizhou Medical University. Funding This study was funded by the National Natural Science Foundation of China (82103681), the Guizhou Medical University National Natural Science Foundation Cultivation Project (grant numbers 20NSP020 and 19NSP034), the Guizhou Provincial Science and Technology Projects (No. ZK [2024]159) and Postgraduate Research Fund of Guizhou Province (No. YJSKYJJ [2021] 143), the Continuous Support Fund for Excellent Scientific Research Platform of Colleges and Universities in Guizhou Province (QJJ (2022) 020), Discipline Leading Talents Project of the Affiliated Hospital of Guizhou Medical University (No.gyfyxkyc-2023-01). Data Availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Klein AP. Pancreatic cancer epidemiology: understanding the role of lifestyle and inherited risk factors. Nat Rev Gastroenterol Hepatol. 2021;18(7):493-502. Park W, Chawla A, O'Reilly EM. Pancreatic Cancer: A Review. JAMA. 2021; 326(9):851-862. Collisson EA, Bailey P, Chang DK, Biankin AV. Molecular subtypes of pancreatic cancer. Nat Rev Gastroenterol Hepatol. 2019; 16(4):207-220. Ransohoff JD, Wei Y, Khavari PA. The functions and unique features of long intergenic non-coding RNA. Nat Rev Mol Cell Biol. 2018; 19(3):143-157. Kopp F, Mendell JT. Functional Classification and Experimental Dissection of Long Noncoding RNAs. Cell. 2018; 172(3):393-407. Ransohoff JD, Wei Y, Khavari PA. The functions and unique features of long intergenic non-coding RNA. Nat Rev Mol Cell Biol. 2018; 19(3):143-157. Bradley RK, Anczuków O. RNA splicing dysregulation and the hallmarks of cancer. Nat Rev Cancer. 2023; 23(3):135-155. Zhang Y, Qian J, Gu C, Yang Y. Alternative splicing and cancer: a systematic review. Signal Transduct Target Ther. 2021;6(1):78. Baralle FE, Giudice J. Alternative splicing as a regulator of development and tissue identity. Nat Rev Mol Cell Biol. 2017; 18(7):437-451. Naro C, Antonioni A, Medici V, Caggiano C, Jolly A, de la Grange P, et al. Splicing targeting drugs highlight intron retention as an actionable vulnerability in advanced prostate cancer. J Exp Clin Cancer Res. 2024; 43(1):58. Ule J, Blencowe BJ. Alternative Splicing Regulatory Networks: Functions, Mechanisms, and Evolution. Mol Cell. 2019;76(2):329-345. Sciarrillo R, Wojtuszkiewicz A, Assaraf YG, Jansen G, Kaspers GJL, Giovannetti E, et al. The role of alternative splicing in cancer: From oncogenesis to drug resistance. Drug Resist Updat. 2020; 53:100728. Chan YT, Lu Y, Wu J, Zhang C, Tan HY, Bian ZX, et al. CRISPR-Cas9 library screening approach for anti-cancer drug discovery: overview and perspectives. Theranostics. 2022 Apr 11;12(7):3329-3344. Xu WW, Liao L, Dai W, Zheng CC, Tan XP, He Y, et al. Genome-wide CRISPR/Cas9 screening identifies a targetable MEST-PURA interaction in cancer metastasis. EBioMedicine. 2023 Jun;92: 104587. Joung J, Engreitz JM, Konermann S, Abudayyeh OO, Verdine VK, Aguet F, et al. Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood. Nature. 2017 Aug 17;548(7667):343-346. Hassn Mesrati M, Syafruddin SE, Mohtar MA, Syahir A. CD44: A Multifunctional Mediator of Cancer Progression. Biomolecules. 2021; 11(12):1850. Yan Y, Zuo X, Wei D. Concise Review: Emerging Role of CD44 in Cancer Stem Cells: A Promising Biomarker and Therapeutic Target. Stem Cells Transl Med. 2015; 4(9):1033-43. Luo H, Li J, Lin Q, Xiao X, Shi Y, Ye X, et al. Ultrasonic irradiation and SonoVue microbubbles-mediated RNA interference targeting PRR11 inhibits breast cancer cells proliferation and metastasis, but promotes apoptosis. Biosci Rep. 2020 ;40(11): BSR20201854. Joung J, Engreitz JM, Konermann S, Abudayyeh OO, Verdine VK, Aguet F, et al. Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood. Nature. 2017 Aug 17;548(7667):343-346. Zeng Z, Lei S, Wang J, Yang Y, Lan J, Tian Q, et al. A novel hypoxia-driven gene signature that can predict the prognosis of hepatocellular carcinoma. Bioengineered. 2022;13(5):12193-12210. Feng Z, Li K, Qin K, Liang J, Shi M, Ma Y, et al. The LINC00623/NAT10 signaling axis promotes pancreatic cancer progression by remodeling ac4C modification of mRNA. J Hematol Oncol. 2022;15(1):112. Jin B, Zhu J, Pan T, Yang Y, Liang L, Zhou Y, et al. MEN1 is a regulator of alternative splicing and prevents R-loop-induced genome instability through suppression of RNA polymerase II elongation. Nucleic Acids Res. 2023;51(15):7951-7971. Taniue K, Akimitsu N. The Functions and Unique Features of LncRNAs in Cancer Development and Tumorigenesis. Int J Mol Sci. 2021;22(2):632. Eptaminitaki GC, Stellas D, Bonavida B, Baritaki S. Long non-coding RNAs (lncRNAs) signaling in cancer chemoresistance: From prediction to druggability. Drug Resist Updat. 2022; 65:100866. Deng L, Liao L, Zhang YL, Hu SY, Yang SY, Ma XY, et al. MYC-driven U2SURP regulates alternative splicing of SAT1 to promote triple-negative breast cancer progression. Cancer Lett. 2023; 560:216124. Nameki N, Takizawa M, Suzuki T, Tani S, Kobayashi N, Sakamoto T, et al. Structural basis for the interaction between the first SURP domain of the SF3A1 subunit in U2 snRNP and the human splicing factor SF1. Protein Sci. 2022;31(10): e4437. De Maio A, Yalamanchili HK, Adamski CJ, Gennarino VA, Liu Z, Qin J, et al. RBM17 Interacts with U2SURP and CHERP to Regulate Expression and Splicing of RNA-Processing Proteins. Cell Rep. 2018;25(3):726-736.e7. Marasco LE, Kornblihtt AR. The physiology of alternative splicing. Nat Rev Mol Cell Biol. 2023;24(4):242-254. Liu Y, Liu X, Lin C, Jia X, Zhu H, Song J, et al. Noncoding RNAs regulate alternative splicing in Cancer. J Exp Clin Cancer Res. 2021;40(1):11. Artemaki PI, Kontos CK. Alternative Splicing in Human Physiology and Disease. Genes (Basel). 2022;13(10):1820. Tao Y, Zhang Q, Wang H, Yang X, Mu H. Alternative splicing and related RNA binding proteins in human health and disease. Signal Transduct Target Ther. 2024;9(1):26. Zhang H, Brown RL, Wei Y, Zhao P, Liu S, Liu X, et al. CD44 splice isoform switching determines breast cancer stem cell state. Genes Dev. 2019;33(3-4):166-179. Bhattacharya R, Mitra T, Ray Chaudhuri S, Roy SS. Mesenchymal splice isoform of CD44 (CD44s) promotes EMT/invasion and imparts stem-like properties to ovarian cancer cells. J Cell Biochem. 2018;119(4):3373-3383. Guo L, Ke H, Zhang H, Zou L, Yang Q, Lu X, et al. TDP43 promotes stemness of breast cancer stem cells through CD44 variant splicing isoforms. Cell Death Dis. 2022;13(5):428. Giraud J, Seeneevassen L, Rousseau B, Bouriez D, Sifré E, Giese A, et al. CD44v3 is a marker of invasive cancer stem cells driving metastasis in gastric carcinoma. Gastric Cancer. 2023;26(2):234-249. Zhu H, Zhou W, Wan Y, Lu J, Ge K, Jia C. CD44V3, an Alternatively Spliced Form of CD44, Promotes Pancreatic Cancer Progression. Int J Mol Sci. 2022;23(20):12061. Liu YN, Tsai MF, Wu SG, Chang TH, Shih JY. CD44s and CD44v8-10 isoforms confer acquired resistance to osimertinib by activating the ErbB3/STAT3 signaling pathway. Life Sci. 2024; 336:122345. Chew SH, Okazaki Y, Akatsuka S, Wang S, Jiang L, Ohara Y, et al. Rheostatic CD44 isoform expression and its association with oxidative stress in human malignant mesothelioma. Free Radic Biol Med. 2017; 106:91-99. Li Z, Chen K, Jiang P, Zhang X, Li X, Li Z. CD44v/CD44s expression patterns are associated with the survival of pancreatic carcinoma patients. Diagn Pathol. 2014;9:79. Gerstberger S, Jiang Q, Ganesh K. Metastasis. Cell. 2023;186(8):1564-1579. Fares J, Fares MY, Khachfe HH, Salhab HA, Fares Y. Molecular principles of metastasis: a hallmark of cancer revisited. Signal Transduct Target Ther. 2020;5(1):28. Ma WK, Voss DM, Scharner J, Costa ASH, Lin KT, Jeon HY, et al. ASO-Based PKM Splice-Switching Therapy Inhibits Hepatocellular Carcinoma Growth. Cancer Res. 2022;82(5):900-915. Ramasamy T, Ruttala HB, Munusamy S, Chakraborty N, Kim JO. Nano drug delivery systems for antisense oligonucleotides (ASO) therapeutics. J Control Release. 2022; 352:861-878. He K, Barsoumian HB, Puebla-Osorio N, Hu Y, Sezen D, Wasley MD, et al. Inhibition of STAT6 with Antisense Oligonucleotides Enhances the Systemic Antitumor Effects of Radiotherapy and Anti-PD-1 in Metastatic Non-Small Cell Lung Cancer. Cancer Immunol Res. 2023;11(4):486-500. Additional Declarations (Not answered) Supplementary Files TableS3.docx Table S3 FigureS4.jpg Figure S4 FigureS3.jpg Figure S3 FigureS1.jpg Figure S1 FigureS2.jpg Figure S2 FigureS5.jpg Figure S5 GraphicalAbstractImage.jpg Graphical Abstract Image TableS4.xlsx Table S4 TableS6.docx Table S6 TableS1.xlsx Table S1 FigureS7.jpg Figure S7 FigureS6.jpg Figure S6 TableS5.xlsx Table S5 TableS2.xlsx Table S2 OriginalImagesforBlots.pdf Original Images for Blots supplementaryfilelegends.docx Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-5388928","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":391379510,"identity":"34b6be79-cad3-4217-973e-5f8ceb802b13","order_by":0,"name":"Shan Lei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYBACPgbGBmYgLQfhshGhhQ2qxZgULQwMIC2JDcRrYW9u/lxQcyd9ftjhBwwfyg4z8M9uIKCF52Cb9Ixjz3I33k4zYJxx7jCDxJ0DBLRIJLYx87Adzt04O4eBmbftMIOBRAIBLfIPmz/z/DucbgjS8pcoLRKMDdJAwxPkpYFaGInSwpPYJs3bd9hwg3SawcGec+k8EjcIaOFnP/74M8+3w/Lys5MfPvhRZi3HP4OAFjgwOMDAAEQMPESqBwL5BuLVjoJRMApGwQgDABiiPxZpKH6UAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-3789-4186","institution":"School of Basic Medical Sciences, Guizhou Medical University","correspondingAuthor":true,"prefix":"","firstName":"Shan","middleName":"","lastName":"Lei","suffix":""},{"id":391379511,"identity":"9b3fa132-6ecf-4998-9b3e-19bea73f3332","order_by":1,"name":"Zhixue Zhang","email":"","orcid":"","institution":"School of Basic Medicine, Guizhou Medical University, Guiyang 550009, Guizhou, China","correspondingAuthor":false,"prefix":"","firstName":"Zhixue","middleName":"","lastName":"Zhang","suffix":""},{"id":391379512,"identity":"e6bb0ac6-96da-438f-bdb6-6effe2bf8ec8","order_by":2,"name":"Zhirui Zeng","email":"","orcid":"https://orcid.org/0000-0001-9547-9074","institution":"Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhirui","middleName":"","lastName":"Zeng","suffix":""},{"id":391379513,"identity":"6fb531f2-4a40-41d6-9ac1-b7f06ff4dacb","order_by":3,"name":"Wenpeng Cao","email":"","orcid":"","institution":"School of Basic Medical Sciences, Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wenpeng","middleName":"","lastName":"Cao","suffix":""},{"id":391379514,"identity":"bbcdb19a-03af-4714-bb45-5d547c5ac811","order_by":4,"name":"Yating Sun","email":"","orcid":"","institution":"Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yating","middleName":"","lastName":"Sun","suffix":""},{"id":391379515,"identity":"9e0a4993-617c-41ce-ace9-23088272853d","order_by":5,"name":"Dahuan Li","email":"","orcid":"","institution":"he Affiliated of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dahuan","middleName":"","lastName":"Li","suffix":""},{"id":391379516,"identity":"0809ed6b-e889-4c1c-9cff-7659a1e10130","order_by":6,"name":"Jigang Pan","email":"","orcid":"","institution":"School of Basic Medical Sciences, Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jigang","middleName":"","lastName":"Pan","suffix":""},{"id":391379517,"identity":"6fe052c6-fb19-4aa1-92b1-035c272a027b","order_by":7,"name":"Yingmin Wu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yingmin","middleName":"","lastName":"Wu","suffix":""},{"id":391379518,"identity":"e83d358c-02e4-47e2-8b4b-9789cf1505f4","order_by":8,"name":"Tuo Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Tuo","middleName":"","lastName":"Zhang","suffix":""},{"id":391379519,"identity":"db96bfaa-1648-4bec-8006-3dc14359d514","order_by":9,"name":"Tengxiang Chen","email":"","orcid":"https://orcid.org/0000-0001-6907-1374","institution":"Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tengxiang","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2024-11-04 14:36:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5388928/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5388928/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72288171,"identity":"96639692-0419-45c9-8062-572a121a46e9","added_by":"auto","created_at":"2024-12-24 17:15:26","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":9291904,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation between HNF1A-AS1 expression and clinical characteristics of PC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Flowchart of CRISPRa screening on AsPC-1 cells; \u003cstrong\u003eB.\u003c/strong\u003e A total of 10 genes were overlapped between the data from genome-wide CRISPR/Cas9 screening and RNA-seq of metastatic PC tissues; \u003cstrong\u003eC.\u003c/strong\u003e qRT-PCR analysis of HNF1A-AS1 expression in 80 paired PC and adjacent non-cancerous tissues; \u003cstrong\u003eD.\u003c/strong\u003e Rank-ordered fold changes of HNF1A-AS1 expression in paired samples from high to low;\u003cstrong\u003e E.\u003c/strong\u003eCorrelation analysis of HNF1A-AS1 expression with perineural invasion; \u003cstrong\u003eF.\u003c/strong\u003eCorrelation analysis of HNF1A-AS1 expression with distant metastasis; \u003cstrong\u003eG. \u003c/strong\u003eCorrelation analysis of HNF1A-AS1 expression with lymph node metastasis; \u003cstrong\u003eH. \u003c/strong\u003eISH analysis depicting HNF1A-AS1 expression levels in metastatic tumor tissues, primary tumor tissues, and adjacent non-tumor tissues; \u003cstrong\u003eI.\u003c/strong\u003e Kaplan-Meier survival analysis comparing overall survival between PC cases with high and low HNF1A-AS1 expression, using the log-rank test; \u003cstrong\u003eJ.\u003c/strong\u003e ROC curve illustrating the diagnostic sensitivity and specificity of HNF1A-AS1 in PC; \u003cstrong\u003eK.\u003c/strong\u003e qRT-PCR measurement of HNF1A-AS1 expression levels in high-metastasis and low-metastasis PC cell lines compared to HPDE cells. n.s., not significant, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/0f080c5396f5534b89d34ac3.jpg"},{"id":72286857,"identity":"f0b7027e-a9f2-4eaa-8b28-42f19535d75e","added_by":"auto","created_at":"2024-12-24 17:07:24","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":7677259,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUpregulation of HNF1A-AS1 in PC enhances tumor metastasis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eWound healing assays comparing the migration capabilities of HNF1A-AS1-overexpressing and -knockdown PC cells with control cells; \u003cstrong\u003eB\u003c/strong\u003e. Transwell assays evaluating the invasive ability of HNF1A-AS1-overexpressing and -knockdown PC cells versus control cells; \u003cstrong\u003eC.\u003c/strong\u003e Western blot analysis demonstrating the impact of HNF1A-AS1 on the expression of EMT markers, including E-cadherin, N-cadherin, and Vimentin; \u003cstrong\u003eD. \u003c/strong\u003eRepresentative bioluminescence images and quantification of swollen inguinal lymph node metastases in mice after subcutaneous footpad injection of HNF1A-AS1-overexpressing, -knockdown, or control PC cells (n = 5); \u003cstrong\u003eE.\u003c/strong\u003e Bioluminescence images and quantification of lung metastases in mice following intravenous injection of HNF1A-AS1-overexpressing, -knockdown, or control PC cells (n = 5); \u003cstrong\u003eF. \u003c/strong\u003eBioluminescence images and quantification of liver metastases in mice after intrasplenic injection of HNF1A-AS1-overexpressing, -knockdown, or control PC cells (n = 5). n.s., not significant, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/87df307f53296cb733bd71b1.jpg"},{"id":72288169,"identity":"3e4750c9-ae70-455d-acfd-f3375f5d9d6b","added_by":"auto","created_at":"2024-12-24 17:15:26","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4801956,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eU2SURP is a binding protein and a key effector of HNF1A-AS1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Flowchart depicting the RNA pulldown-MS approach for identifying HNF1A-AS1-binding proteins; \u003cstrong\u003eB.\u003c/strong\u003e KEGG analysis revealing that HNF1A-AS1-binding proteins are involved in the spliceosome; \u003cstrong\u003eC. \u003c/strong\u003eVenn diagram illustrating the identification of CDC5L, SF3B2, HNRNPM, U2SURP, and PCBP1 as HNF1A-AS1-binding proteins based on proteomic data and splicing factor families; \u003cstrong\u003eD-E.\u003c/strong\u003e Interaction between HNF1A-AS1 and U2SURP in PC cells demonstrated by RNA pulldown (D) and RIP-qPCR (E) assays; \u003cstrong\u003eF.\u003c/strong\u003e FISH and immunofluorescence analysis of the co-localisation of HNF1A-AS1 and U2SURP in the nucleus of PC cells; \u003cstrong\u003eG.\u003c/strong\u003eSchematic diagram of HNF1A-AS1 mutation design and the result of gel electrophoresis analysis; \u003cstrong\u003eH.\u003c/strong\u003e Detection of binding between HNF1A-AS1 mutants and U2SURP; \u003cstrong\u003eI.\u003c/strong\u003e Schematic diagram and Western blot analysis of U2SURP truncated mutants; \u003cstrong\u003eJ. \u003c/strong\u003eRIP-qPCR assay showing binding affinity between U2SURP truncated mutants and HNF1A-AS1. n.s., not significant, *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/dfb417383f0e1146112bb39d.jpg"},{"id":72286860,"identity":"fb836046-5a8b-4844-b0c7-e3eabe934626","added_by":"auto","created_at":"2024-12-24 17:07:24","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5066371,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHNF1A-AS1 binds to both U2SURP and CD44 pre-mRNA to enhance their interaction.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eHeatmap displaying PSI values for differentially spliced ASEs between HNF1A-AS1 knockdown and control AsPC-1 cells; \u003cstrong\u003eB.\u003c/strong\u003e Schematic (left) and distribution (right) of five alternative splicing types that change upon HNF1A-AS1 knockdown in AsPC-1 cells; \u003cstrong\u003eC.\u003c/strong\u003e Pie chart illustrating the probability of alternative splicing events affected by HNF1A-AS1; \u003cstrong\u003eD. \u003c/strong\u003eRT-PCR and gel electrophoresis analysis of selected SE genes in HNF1A-AS1 knockdown and control AsPC-1 cells.Target exons are indicated in the left panel: blue box, constitutive exon; red box, skipped exons; \u003cstrong\u003eE.\u003c/strong\u003e MS2-RIP-qPCR assay confirming the binding of HNF1A-AS1 to CD44 pre-mRNA in PC cells; \u003cstrong\u003eF.\u003c/strong\u003e RIP-qPCR assay investigating the binding affinity between U2SURP truncated mutants and CD44; \u003cstrong\u003eG.\u003c/strong\u003eMS2-RIP-qPCR assay monitoring the binding affinity between HNF1A-AS1 truncated mutants and CD44; \u003cstrong\u003eH.\u003c/strong\u003e RIP-qPCR assay assessing the effect of HNF1A-AS1overexpression or knockdown on the U2SURP-CD44 interaction. n.s., not significant, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/c63e2b13a5c6d131a595312a.jpg"},{"id":72286885,"identity":"88d9b1b2-69cc-4b4c-801a-ea94c1f341e3","added_by":"auto","created_at":"2024-12-24 17:07:25","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":7868117,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eU2SURP-induced alternative splicing of CD44 mediates HNF1A-AS1-driven cancer metastasis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. Schematic diagrams of alternative splicing events generating CD44s and CD44v (3-10); \u003cstrong\u003eB-C.\u003c/strong\u003e Expression levels of CD44s and CD44v (3-10) assessed by qRT-PCR and Western blot in PC cells with either HNF1A-AS1 overexpression or knockdown; \u003cstrong\u003eD. \u003c/strong\u003eWestern blot analysis of CD44v (3-10) in PC cells with U2SURP knockdown; \u003cstrong\u003eE.\u003c/strong\u003e qRT-PCR analysis comparing CD44v (3-10) and CD44s expression in 80 paired PC and normal tissues; \u003cstrong\u003eF.\u003c/strong\u003e Expression of CD44v (3-10) in a cohort of 80 primary PC tumors and adjacent normal tissues; \u003cstrong\u003eG-H.\u003c/strong\u003e Kaplan-Meier survival analysis for patients with PC stratified by CD44v (3-10) expression levels; \u003cstrong\u003eI-L\u003c/strong\u003e. Wound healing assay, Transwell assay, and bioluminescence imaging (n = 5) demonstrating that CD44v (3-10) counteracts the inhibitory effects of HNF1A-AS1 knockdown on PC cell invasion and metastasis. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/9519bb61c2319b2a34c0b5ca.jpg"},{"id":72286888,"identity":"f7001919-0910-48fb-881b-4c4655e44313","added_by":"auto","created_at":"2024-12-24 17:07:25","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5607453,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSNAI2 regulates HNF1A-AS1 expression by binding to its promoter.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. Binding motif for SNAI2 predicted by JASPAR; \u003cstrong\u003eB.\u003c/strong\u003e Diagram showing potential SNAI2 binding sites within the HNF1A-AS1 promoter region; \u003cstrong\u003eC.\u003c/strong\u003eDiagram of primers designed to target regions of the HNF1A-AS1 promoter; \u003cstrong\u003eD. \u003c/strong\u003eChIP-PCR analysis demonstrating SNAI2 enrichment at the HNF1A-AS1 promoter, with IgG as a negative control; \u003cstrong\u003eE. \u003c/strong\u003eConstruction of dual-luciferase reporter vectors for the HNF1A-AS1 promoter; \u003cstrong\u003eF.\u003c/strong\u003eLuciferase activity assays of wild-type and mutant HNF1A-AS1 promoters in indicated cells; \u003cstrong\u003eG.\u003c/strong\u003e RT-qPCR analysis of HNF1A-AS1 expression in PC cells with either SNAI2 overexpression or knockdown; \u003cstrong\u003eH.\u003c/strong\u003e FISH and immunofluorescence analysis of the co-localisation of SNAI2, HNF1A-AS1, and U2SURP in PC tissue samples. **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/9c49743d8f3ad16f8e7d9cfa.jpg"},{"id":72288186,"identity":"d3df3525-5d0c-4546-b40f-7a8a38069c4c","added_by":"auto","created_at":"2024-12-24 17:15:28","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":8637484,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eASO inhibits PC cell metastasis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Relative expression levels of HNF1A-AS1 in PC cells with or without ASO treatment; \u003cstrong\u003eB.\u003c/strong\u003eTranswell assays assessing the invasive capabilities of PC cells treated with ASO1-HNF1A-AS1 or ASO-NC; \u003cstrong\u003eC.\u003c/strong\u003e Wound healing assays evaluating the migration abilities of PC cells treated with ASO1-HNF1A-AS1 or ASO-NC; \u003cstrong\u003eD. \u003c/strong\u003eWestern blot analysis showing the impact of ASO1-HNF1A-AS1 on CD44v (3-10) and EMT marker expression levels; \u003cstrong\u003eE.\u003c/strong\u003e Flowchart illustrating the metastasis model and treatment protocol in mice;\u003cstrong\u003e F.\u003c/strong\u003e Bioluminescence imaging and quantification demonstrating the effect of ASO1-HNF1A-AS1 or ASO-NC on liver metastasis in mice; \u003cstrong\u003eG.\u003c/strong\u003e Bioluminescence imaging and quantification showing the impact of ASO1-HNF1A-AS1 or ASO-NC on lung metastasis in mice;\u003cstrong\u003e H.\u003c/strong\u003e Bioluminescence imaging and quantification revealing the effect of ASO1-HNF1A-AS1 or ASO-NC on lymph node metastasis in mice.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/ab72ed7e26dc43a57454ebfb.jpg"},{"id":72901297,"identity":"b953713e-936a-4c39-b6df-41f5d4d03122","added_by":"auto","created_at":"2025-01-03 12:49:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":49730611,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/9e384b7c-e590-4af3-bc4f-d083ef714eea.pdf"},{"id":72286841,"identity":"0f4e5e64-ffdc-4cb4-b9c6-d00083b6a312","added_by":"auto","created_at":"2024-12-24 17:07:23","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19140,"visible":true,"origin":"","legend":"Table S3","description":"","filename":"TableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/42e237669cc62706ef2c1d5e.docx"},{"id":72286850,"identity":"11adda7c-f6cb-4b9e-8a7f-f4d52c97f664","added_by":"auto","created_at":"2024-12-24 17:07:24","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5366698,"visible":true,"origin":"","legend":"Figure S4","description":"","filename":"FigureS4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/560104982939a665f292be7a.jpg"},{"id":72286849,"identity":"e61bde4d-c4f3-4f59-8e69-970f8b4c6546","added_by":"auto","created_at":"2024-12-24 17:07:24","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2208734,"visible":true,"origin":"","legend":"Figure S3","description":"","filename":"FigureS3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/0c784093b9bf8121ac258f3b.jpg"},{"id":72286861,"identity":"5b60ec95-0a48-4d28-8eca-ffa2a595300f","added_by":"auto","created_at":"2024-12-24 17:07:24","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3935842,"visible":true,"origin":"","legend":"Figure S1","description":"","filename":"FigureS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/f6a5ae3c329230aa89a5a8dc.jpg"},{"id":72288178,"identity":"91594230-9626-4d10-b128-52840cbdcb56","added_by":"auto","created_at":"2024-12-24 17:15:27","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":846633,"visible":true,"origin":"","legend":"Figure S2","description":"","filename":"FigureS2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/d8e3395aa11f86ee5e874e90.jpg"},{"id":72286853,"identity":"0830c4f5-2bdb-4ce7-8a83-fba14990d144","added_by":"auto","created_at":"2024-12-24 17:07:24","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":2120473,"visible":true,"origin":"","legend":"Figure S5","description":"","filename":"FigureS5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/b79d87e02bda42545afff984.jpg"},{"id":72286846,"identity":"1c3bc69e-a5bb-4db3-99ca-36755c652339","added_by":"auto","created_at":"2024-12-24 17:07:24","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":112786,"visible":true,"origin":"","legend":"Graphical Abstract Image","description":"","filename":"GraphicalAbstractImage.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/c95ba09d065eedcdf601d22b.jpg"},{"id":72288173,"identity":"c30e0f99-8e87-41dc-bff3-7f88b9ea2088","added_by":"auto","created_at":"2024-12-24 17:15:27","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":28868,"visible":true,"origin":"","legend":"Table S4","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/8b7cff9881b0254ce7e19cfc.xlsx"},{"id":72286851,"identity":"babf16ff-773e-4a54-9377-7ea53b069909","added_by":"auto","created_at":"2024-12-24 17:07:24","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":19460,"visible":true,"origin":"","legend":"Table S6","description":"","filename":"TableS6.docx","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/40b0cc8344018850f99995ac.docx"},{"id":72288182,"identity":"bd745abc-254f-4d36-b391-5920213ba541","added_by":"auto","created_at":"2024-12-24 17:15:28","extension":"xlsx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":12565,"visible":true,"origin":"","legend":"Table S1","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/33c9a5bc6bbc39bc83d00598.xlsx"},{"id":72286876,"identity":"0ea27230-7611-4b7e-a8e6-72277edd3981","added_by":"auto","created_at":"2024-12-24 17:07:25","extension":"jpg","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":1258064,"visible":true,"origin":"","legend":"Figure S7","description":"","filename":"FigureS7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/58897ce5c1eb3a58c29a6a3a.jpg"},{"id":72286863,"identity":"bee37198-2075-4008-8e59-72cd296948ff","added_by":"auto","created_at":"2024-12-24 17:07:25","extension":"jpg","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":2302363,"visible":true,"origin":"","legend":"Figure S6","description":"","filename":"FigureS6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/b9a897e6da74cb681d490ae1.jpg"},{"id":72286854,"identity":"e624340e-8fdc-4b91-a3f3-51729b2749ef","added_by":"auto","created_at":"2024-12-24 17:07:24","extension":"xlsx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":3353427,"visible":true,"origin":"","legend":"Table S5","description":"","filename":"TableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/c9d6cd576b11dad22484dd72.xlsx"},{"id":72286879,"identity":"cd897c8a-3cb7-42c8-bfa2-69221d007f43","added_by":"auto","created_at":"2024-12-24 17:07:25","extension":"xlsx","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":19730,"visible":true,"origin":"","legend":"Table S2","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/84351fcf350d8efdf0480f4a.xlsx"},{"id":72286852,"identity":"c744c7fd-4dd7-4a1d-879b-ef68a50c84ec","added_by":"auto","created_at":"2024-12-24 17:07:24","extension":"pdf","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":2068858,"visible":true,"origin":"","legend":"Original Images for Blots","description":"","filename":"OriginalImagesforBlots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/080b930c204140948bc89524.pdf"},{"id":72286855,"identity":"3c42e840-1767-421e-bc57-32b40080b771","added_by":"auto","created_at":"2024-12-24 17:07:24","extension":"docx","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":14965,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfilelegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-5388928/v1/d07f41559dbaf854f04aa261.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Genome-scale activation screen reveals lncRNA HNF1A-AS1 as a novel therapeutic target for pancreatic cancer metastasis","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePancreatic cancer (PC) ranks as the fourth leading cause of cancer-related mortality globally. Despite advances in treatment, the 5-year survival rate for patients with PC remains below 5% [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The poor prognosis is largely due to the early invasion and rapid proliferation of cancer cells [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Thus, it is crucial to understand the molecular mechanisms underlying PC development and to develop effective therapeutic strategies.\u003c/p\u003e \u003cp\u003eLong non-coding RNAs (LncRNAs), which exceed 200 nucleotides in length but lack coding potential, have been implicated in various biological processes, including the cell cycle, apoptosis, aging, metastasis, and chemotherapy resistance [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Recent research highlights the pivotal role of alternative splicing (AS) in tumorigenesis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. AS generates multiple RNA transcripts from a single precursor mRNA, offering a promising area for enhancing clinical diagnosis, disease prediction, and therapeutic development [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Dysregulation of AS can lead to diseases and impact gene stability, with significant implications for tumor biology [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, identifying and understanding AS-related genes in PC may provide valuable insights for effective treatment.\u003c/p\u003e \u003cp\u003eGiven the noncoding nature and diverse functionalities of long noncoding RNAs (lncRNAs), a systematic methodology is imperative to elucidate their roles in prostate cancer (PC) metastasis. Clustered regularly interspaced short palindromic repeats (CRISPR) coupled with CRISPR-associated protein 9 (Cas9) screening represents a novel technique for identifying potential essential genes or therapeutic targets through gene knockout or activation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. A CRISPR library comprises thousands of single-guide RNAs (sgRNAs), which can target both protein-coding genes and noncoding RNAs. Through a knockout strategy, essential genes can be discerned via negative selection [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Another CRISPR/Cas9 strategy developed by Zhang employs a three-component engineered protein complex capable of activating the transcription of long non-coding RNAs (lncRNAs) via single guide RNAs (sgRNAs) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Utilizing a lncRNA activation library, genes that inhibit cancer metastasis can be identified through negative selection, whereas genes that drive cancer metastasis are prevalent in the positive selection results. This powerful tool facilitates the elucidation of the molecular mechanisms underlying prostate cancer (PC) metastasis and other cancer types, allowing for the consideration of previously undiscovered targets.\u003c/p\u003e \u003cp\u003eCD44, a multifunctional transmembrane glycoprotein, is essential for maintaining normal cellular functions and has been shown to influence cancer development and progression [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. CD44 undergoes alternative splicing to produce various isoforms with distinct functions [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, the molecular mechanisms driving AS and the roles of CD44 variants in cancer remain poorly understood. This study compared CD44v (3\u0026ndash;10) and CD44s interactomes using mass spectrometry (MS)-based proteomics. It investigated the biological role and clinical relevance of HNF1A-AS1 in cancer metastasis, examining its potential regulation of CD44 alternative splicing through U2SURP. Additionally, the study explored how CD44 variants contribute to cancer metastasis and assessed an antisense oligonucleotide (ASO) targeting HNF1A-AS1 in preclinical trials for its potential to inhibit cancer metastasis.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNormal human pancreatic epithelial cells (HPDE) and PC cell lines (BxPC-3, PANC-1, MIA PaCa-2, AsPC-1, PATU8988-S, Capan-1) were procured from DSMZ (Braunschweig, Germany). The PANC-1-Luc and AsPC-1-Luc cell lines were obtained from Procell (Wuhan, China). PANC-1 and MIA PaCa-2 cells were cultured in DMEM (Dulbecco\u0026apos;s Modified Eagle Medium; Gibco, USA) with 10% FBS (Bioind, Israel), while HPDE, AsPC-1, Capan-1, PATU8988-S, and BxPC-3 cells were maintained in RPMI-1640 (Roswell Park Memorial Institute-1640 Medium; Gibco, USA) with 10% FBS. All cell lines were incubated at 37\u0026deg;C with 5% CO2 and verified by short tandem repeat profiling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid construction and cell transfection plasmids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLentiviruses for HNF1A-AS1 knockdown, overexpression, and negative controls were sourced from Genechem (Shanghai, China). Transfections of plasmids, siRNAs, or ASOs were performed using Lipofectamine 3000 (Thermo Fisher Scientific) as per the manufacturer\u0026apos;s protocol [18]. The pcDNA3.1 vector (Addgene plasmid 52961) was used to clone CD44s, CD44v (3-10), and U2SURP coding sequences. Mutants were generated using primers with mismatched nucleotides. HNF1A-AS1 ASOs were provided by GenePharma (Shanghai, China). All sequences used in this study are summarized in Supplementary Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;CRISPR‒Cas9 library screen\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCRISPR-Cas9 long non-coding RNA (lncRNA) activation screens were conducted on pooled human CRISPR 3-plasmid lncRNA Synergistic Activation Mediator (SAM) libraries (Addgene #1000000106). The library contains 95,058 single guide RNAs (sgRNAs) targeting 10,504 lncRNAs, with around ten sgRNAs aimed at the transcription start site (TSS) of each lncRNA. Using Endura electrocompetent cells, the sgRNA library was cloned according to protocol and amplified according to the manufacturer\u0026apos;s instructions [19]. AsPC-1 cells were transduced with dCas9-VP64 and MPHv2 and subjected to a five-day selective selection regime using the purified sgRNA library and the other two components of the SAM system: dCas9-VP64-blast and MS2-P65-HSF1. After zeocin kill curve analysis, stable clones were transduced with a sgRNA library at a multiplicity of infection (MOI) less than 0.3, ensuring that each cell only contained one sgRNA. Approximately 2\u0026times;10\u003csup\u003e7\u003c/sup\u003e cells were harvested one week later to collect input DNA, with those remaining cells selected using Matrigel covered Boyden chambers with 300 g/mL zeocin (InvivoGen, USA). A sufficient number of cells were obtained for the next invasion selection round by removing invading and adhering cells with trypsin and culturing them. Reseeding was then performed three times in new invasion chambers. In order to ensure adequate representation of sgRNA, a minimum of 7\u0026times;10\u003csup\u003e7\u003c/sup\u003e cells were maintained at all times. To extract total genomic DNA, the Zymo Research Quick-gDNA MidiPrep Kit (Zymo Research, USA) was used. After the third round of screening, the cells were harvested and genomic DNA was extracted. In a subsequent PCR, ten pairs of barcoded primers were used to amplify the subregions targeted by the sgRNA. The PCR products were purified using Qiagen gel kits and then analyzed for sgRNAs via next-generation paired-end sequencing (Novogene).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR and Western blotting analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWestern blotting and qRT-PCR were conducted as previously described [20], with qRT-PCR reagents supplied by Takara (Japan). Primer sequences for qRT-PCR and antibodies for Western blotting are detailed in Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue microarray, in situ hybridization (ISH), and immunohistochemistry (IHC) staining \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemical staining for CD44v (3-10) followed established protocols [21]. Briefly, tissue samples were deparaffinized in xylene, rehydrated through a gradient of alcohols, and subjected to high-temperature antigen retrieval. Sections were then incubated overnight at 4\u0026deg;C with antibodies against CD44v (3-10) and vimentin, followed by biotinylated secondary antibodies. Visualization was achieved using DAB (3,3\u0026apos;-Diaminobenzidine; Dako, Mississauga, ON, Canada).\u0026nbsp;HNF1A-AS1 in situ hybridization (ISH) probes were obtained from Qiagen (Hilden, Germany), and ISH was performed according to the instructions provided with the ISH kitTM (Servicebio, Wuhan, China). Tissue microarrays were deparaffinized in xylene, rehydrated with ethanol, and digested with 20 \u0026micro;g/ml pepsin. After hybridization with digoxigenin-labeled probes and secondary antibodies, samples were stained with DAB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA pulldown\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MegaScript\u003csup\u003eTM\u003c/sup\u003e T7 Transcription Kit (Invitrogen) was utilized to purify truncated mutations or antisense RNA by transcribing full-length or truncated variants with T7 RNA polymerase (Roche Diagnostics, Indianapolis, USA). Biotin-labeled lncRNA was captured using streptavidin magnetic beads and incubated with PC cell lysate at 4\u0026deg;C for 1 hour. Eluates were then analyzed by mass spectrometry or Western blotting following separation and purification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMS2-RIP-MS/qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003epcDNA3.1 constructs including 24 \u0026times; MS2-HNF1A-AS1, 24 \u0026times; MS2 empty vector, 24 \u0026times; MS2-antisense-HNF1A-AS1, or 24 \u0026times; MS2-HNF1A-AS1 mutants (△BR1,\u0026nbsp;△BR2,\u0026nbsp;△BR3,\u0026nbsp;△BR4,\u0026nbsp;△BR5) were co-transfected with pMS2-GST. Cells were harvested 48 hours post-transfection, and RNA-binding proteins were purified using the Magna RIP RNA Binding Protein Immunoprecipitation Kit (Millipore) for qRT-PCR analysis. MS was used to identify proteins interacting with sense and antisense HNF1A-AS1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranswell invasion assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInvasion assays were conducted as previously described [22]. Matrigel (20 \u0026micro;g; BD Biosciences) was evenly applied to the upper chamber membrane. Subsequently, 2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells resuspended in 200 \u0026micro;L of serum-free medium were seeded into the upper chamber. The lower chamber was filled with 700 \u0026micro;L of medium containing 10% FBS. After 24 hours of incubation, cells were fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 20 minutes, washed, and observed under an inverted microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWound healing assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor migration assays, PC cells from different treatment groups were seeded in six-well plates and grown to approximately 100% confluence. Cells were scraped with a 200 \u0026micro;L pipette tip, and serum-containing media were removed. The cultures were then treated with mitomycin-C and incubated for 24 hours. Migration was assessed by capturing images at 0 and 24 hours post-scratching.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLymph node metastasis model assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLuciferase-expressing PC cells (2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per mouse) suspended in 150 \u0026micro;L of PBS were inoculated subcutaneously into the left hind foot pad of 6 to 8-week-old female immunodeficient mice. Mice were randomly assigned to treatment groups (n = 5 per group). Following intraperitoneal injection of 100 \u0026micro;L of D-luciferin methyl salt (150 mg/kg in PBS), mice were anesthetized with 3% isoflurane gas and monitored for tumor metastasis using the IVIS 100 imaging system (Caliper Life Sciences, Waltham, MA, USA). Mice were then euthanized, and tumors and lymph nodes were excised, fixed in 4% PFA, and subjected to histological analysis. Treatments were administered in a non-blinded manner. All animal experiments were conducted following the guidelines of the Animal Ethics Committee at Guizhou Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;metastasis model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe splenic injection assay assessed the liver metastasis capability of various PC cell groups. A total of 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e luciferase-expressing PC cells in 100 \u0026micro;L PBS were injected into the spleens of 6-week-old female BALB/C nude mice (n = 5 per group). For evaluating lung\u0026nbsp;metastasis, the same PC cells were administered \u003cem\u003evia\u003c/em\u003e tail vein injection into female BALB/C nude mice (n = 5 per group, 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per mouse). Mice were weighed weekly and euthanized after 12 weeks. Following tail vein injection, 100 \u0026micro;L of D-luciferin methyl salt (150 mg/kg in PBS) was intraperitoneally administered, and metastatic spread was monitored using the IVIS 100 Imaging System (Caliper Life Sciences, Waltham, MA, USA). Lungs and livers were excised, fixed in 4% paraformaldehyde for 24-48 hours, embedded in paraffin, and subjected to Hematoxylin-Eosin (H\u0026amp;E) staining to identify metastatic sites.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromatin immunoprecipitation (ChIP)-qPCR assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChIP assays were conducted following the SimpleChIP\u0026reg; Enzymatic Chromatin IP Kit (Magnetic Beads) (9003S, CST) protocol. Cells (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) were cross-linked with 1% formaldehyde at room temperature for 10 minutes. Cell pellets were then incubated in Buffer A at 4\u0026deg;C for 10 minutes before nuclei were isolated by centrifugation at 5000 rpm. Nuclei were subsequently digested in Buffer B with 25 units of micrococcal nuclease (M Nase; 10011, CST) for 20 minutes at 37\u0026deg;C. DNA was fragmented using pulsed ultrasonication, and chromatin was collected by centrifugation at 12,000 rpm for 10 minutes. Chromatin DNA was quantified, and an equal amount was incubated overnight at 4\u0026deg;C with either the target antibody or a negative control IgG. Immunoprecipitated complexes were then incubated with Protein A/G magnetic beads for 2 hours at 4\u0026deg;C, extensively washed, and DNA was purified using spin columns. Quantitative PCR with TB Green Premix Ex Taq\u0026trade; on a CFX Connect Real-Time System was used to quantify the purified DNA. Quantification was based on input and IgG-enriched signals, with values normalized to a baseline of 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRT\u0026ndash;PCR detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor reverse-transcription PCR (RT-PCR) assays, primers were designed using Primer Premier 5 based on alternative splicing event (ASE) analysis to detect exon skipping and validate RNA splicing. The RT-PCR protocol consisted of an initial denaturation at 95\u0026deg;C for 2 minutes, followed by 36 cycles of denaturation at 95\u0026deg;C for 30 seconds, annealing at 56\u0026deg;C for 30 seconds, and elongation at 72\u0026deg;C for 1 minute. A final elongation step was performed at 72\u0026deg;C for 5 minutes, with the reaction held at 4\u0026deg;C. PCR products were separated on 2% agarose gels stained with 0.1\u0026permil; ethidium bromide and visualized using a GleUV system (Baygene Biotech). Band intensities were quantified with ImageJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach \u003cem\u003ein vitro\u003c/em\u003e experiment was replicated three times. Statistical analyses were conducted using GraphPad Prism 8.0 (GraphPad Software Inc., La Jolla, CA, USA) and Student\u0026apos;s t-test. Survival analysis was performed with the Kaplan-Meier method and log-rank test using Statistical Package for the Social Sciences (SPSS) software (SPSS Inc., Chicago, IL, USA). Data are presented as mean \u0026plusmn; S.D. from three independent experiments, with statistical significance determined at a P-value of \u0026lt; 0.05.\u003c/p\u003e"},{"header":"RESULT","content":"\u003cp\u003e\u003cstrong\u003eExpression of HNF1A-AS1 and its relationship to clinical parameters in patients with PC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKey lncRNAs involved in cancer metastasis were identified through RNA sequencing of three metastatic tumor tissues and three primary tumor tissues (Figure S1A). Of the 287 differentially expressed lncRNAs (log2 FC \u0026gt; 1), 146 were downregulated and 140 upregulated in metastatic tissues (Figure S1B, Table S2). We implemented a comprehensive screening strategy utilizing a CRISPR/Cas9 lncRNA SAM pooled library, which comprised 96,458 sgRNAs targeting the transcription start sites (TSSs) of 10,504 distinct lncRNAs [15]. The human pancreatic cancer cell line AsPC-1 served as the in vitro model for CRISPR activation (CRISPRa) screening to identify potential lncRNA regulators. The cell population was bifurcated into two groups: one designated as the control (Input) and the other subjected to three rounds of invasion selection (Cas9) using Matrigel-coated Boyden chambers. Genomic DNA was extracted from both cell populations, and next-generation sequencing was employed to quantify the read counts of each sgRNA (Figure 1A). \u0026nbsp;The Cas9 cells demonstrated an increased invasive capacity relative to the input cells (Figure S1C). Subsequent next-generation sequencing of genomic DNA indicated a substantial reduction in the diversity of sgRNAs within the Cas9 cells, identifying 31 significantly depleted sgRNAs that target 45 genes. Moreover, an integrated analysis combining the 76 genes identified from the CRISPR/Cas9 screening with the 287 differentially expressed lncRNAs in metastatic tumor tissues yielded a shortlist of 10 overlapping lncRNAs (Figure 1B). Analysis of 80 human PC tissues and adjacent noncancerous samples via qRT-PCR detected the expression levels of the 10 lncRNAs, with HNF1A-AS1 showing markedly higher expression in PC tissues compared to adjacent noncancerous tissues, thereby becoming the focal point of this study (Figure 1C, Figure S1D). In 50 of 77 (65%) clinical sample pairs, HNF1A-AS1 expression was significantly elevated in tumor tissues relative to matched adjacent non-tumor tissues (Figure 1D). The correlation between HNF1A-AS1 expression and clinicopathological parameters of PC was further explored, revealing a significant positive association with perineural invasion (Figure 1E), distant metastasis (Figure 1F), and lymph node metastasis (Figure 1G). Other factors, such as gender, age, differentiation grade, tumor site, and tumor size, did not show significant differences (Table S3). In situ hybridization staining confirmed that HNF1A-AS1 levels were substantially higher in PC tumor tissues than in adjacent non-tumor tissues (Figure 1H). Patients were categorized into high and low HNF1A-AS1 expression groups based on median expression levels. Kaplan-Meier survival analysis, supplemented by the log-rank test, indicated that patients with lower HNF1A-AS1 expression exhibited improved overall survival compared to those with higher levels (Figure 1I). ROC curve analysis of 80 paired tissue samples yielded an area under the curve (AUC) of 0.8786, suggesting HNF1A-AS1\u0026apos;s potential as a diagnostic marker for PC (Figure 1J). Additionally, qRT-PCR results demonstrated significantly higher HNF1A-AS1 expression in PC cell lines with high metastatic potential (AsPC-1, Capan-1, PATU8988-S) compared to those with lower metastatic capability (BxPC-3, PANC-1, MIA PaCa-2) and human pancreatic epithelial immortalized cells (HPDE) (Figure 1K).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUpregulation of HNF1A-AS1 in PC promotes cancer metastasis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the biological role of HNF1A-AS1, low-metastasis PC cell lines (BxPC-3, PANC-1) with HNF1A-AS1 overexpression and high-metastasis PC cell lines (AsPC-1, Capan-1) with HNF1A-AS1 knockdown were developed (Figure 2S). Wound-healing assays demonstrated that HNF1A-AS1 overexpression enhanced PC cell migration, while HNF1A-AS1 knockdown significantly impeded migration (Figure 2A). Transwell invasion assays confirmed that overexpression of HNF1A-AS1 promoted invasion, whereas its knockdown reduced invasive capabilities (Figure 2B). Analysis of EMT marker expression revealed decreased E-cadherin levels and increased vimentin and N-cadherin levels, which facilitated cell migration and invasion (Figure 2C). Additionally, popliteal lymph node metastasis models indicated that HNF1A-AS1 overexpression resulted in more pronounced lymphadenopathy compared to controls, while knockdown led to reduced lymphadenopathy (Figure 2D). \u003cem\u003eIn vivo\u003c/em\u003e bioluminescence imaging showed stronger signals in nude mice intravenously injected with HNF1A-AS1 overexpressing PC cells, in contrast to those with knockdown (Figure 2E). Similarly, liver bioluminescence signals were more intense in mice subjected to splenic injection of HNF1A-AS1 overexpressing PC cells, whereas knockdown resulted in diminished signals (Figure 2F).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHNF1A-AS1 interacts with U2SURP to exert its biological function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe subcellular localization of HNF1A-AS1 was examined through nuclear-cytoplasmic fractionation and FISH assays, revealing HNF1A-AS1 is mainly localized in the nucleus (Figure S3A-B). To elucidate the molecular mechanisms underlying HNF1A-AS1\u0026apos;s role in cancer metastasis, RNA pulldown assays coupled with MS analysis were conducted to identify its binding proteins (Figure 3A, Table S4). KEGG pathway analysis highlighted the spliceosome signaling pathway as significantly enriched among the associated proteins (Figure 3B). Integration with the splicing factor family identified CDC5L, SF3B2, HNRNPM, U2SURP, and PCBP1 as key candidates for further investigation (Figure 3C). RNA pull-down assays confirmed the binding of HNF1A-AS1 to these proteins, with U2SURP showing the most significant interaction in Capan-1 and AsPC-1 cells (Figure 3D, Figure S3C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRIP assays further validated the enrichment of HNF1A-AS1 in U2SURP immunoprecipitates (Figure 3E), and co-localization of HNF1A-AS1 with U2SURP was observed in PC cells (Figure 3F). To pinpoint the regions of HNF1A-AS1 responsible for binding U2SURP, a series of deletion mutants were constructed: △BR1 (1-500 nt), △BR2 (501-1000 nt), △BR3 (1001-1500 nt), △BR4 (1501-2000 nt), and △BR5 (2001-2455 nt) (Figure 3G). RNA pulldown assays demonstrated that binding to U2SURP is confined to the 1001-1500 nt region (Figure 3H). Further analysis using truncated binding motif mutants, including △RRM and △CID, revealed that deletion of the RRM domain disrupted the HNF1A-AS1-U2SURP interaction (Figure 3I-J), underscoring the critical role of the RRM region in this binding association. Subsequently, investigations into U2SURP\u0026apos;s role in PC metastasis were initiated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHNF1A-AS1 acts as a scaffold to promote the binding of U2SURP to CD44 pre-mRNA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunctional assays demonstrated that U2SURP-deficient impairs the ability of invasion and metastasis of pancreatic cancer cells overexpressing HNF1A-AS1 (Figure S4A-B), underscoring U2SURP\u0026apos;s critical role in facilitating cancer metastasis through HNF1A-AS1. Subsequent investigations into the HNF1A-AS1-U2SURP interaction revealed that modifying HNF1A-AS1 did not impact U2SURP\u0026apos;s expression levels (Figure S4C). Given U2SURP\u0026apos;s role as a splicing factor, it was hypothesized that HNF1A-AS1 might modulate U2SURP-mediated alternative mRNA splicing. To assess HNF1A-AS1\u0026apos;s involvement in AS, deep RNA-seq was performed on HNF1A-AS1 knockdown AsPC-1 cells, followed by rMATS analysis of splicing events (Figure 4A). The analysis revealed significant changes in AS profiles post-HNF1A-AS1 knockdown, identifying 1593 ASEs across 1451 genes (Table S5). The majority of ASEs were skipped exons (61.9%), followed by alternative 3\u0026apos; splice sites (14%), mutually exclusive exons (12.68%), alternative 5\u0026apos; splice sites (8.1%), and retained introns (3.4%) (Figure 4B). Notably, TIMMDC1, RER1, PSMA4, and HROB emerged as top candidates for alternative splicing, suggesting that HNF1A-AS1 may act as a scaffold to influence U2SURP binding to these targets. PCR primer pairs were designed to target adjacent constitutive exons in genes with skipped exons regulated by HNF1A-AS1.\u0026nbsp;HNF1A-AS1 knockdown in AsPC-1 and Capan-1 cells\u0026nbsp;resulted in increased\u0026nbsp;in exon 7 skipping of CD44\u0026nbsp;(Figure 4D, Figure S4D), indicating that HNF1A-AS1 might modulate splicing by acting as a scaffold for U2SURP. MS2-RIP-qPCR assays confirmed that HNF1A-AS1 interacts with CD44 pre-mRNA but not with TIMMDC1, RER1, PSMA4, or HROB (Figure 4E, Figure S5), suggesting CD44 as a potential downstream target of HNF1A-AS1.\u003c/p\u003e\n\u003cp\u003eRIP-qPCR assays further demonstrated that U2SURP binds to CD44 pre-mRNA, with the RRM domain of U2SURP essential for this interaction (Figure 4F). MS2-RIP-qPCR experiments, employing full-length and truncated HNF1A-AS1 fragments (△BR1,\u0026nbsp;△BR2,\u0026nbsp;△BR3,\u0026nbsp;△BR4,\u0026nbsp;△BR5), pinpointed the BR3 region as crucial for HNF1A-AS1\u0026apos;s binding to CD44 pre-mRNA (Figure 4G). Additionally, gain- and loss-of-function experiments, combined with RNA immunoprecipitation using an anti-U2SURP antibody, demonstrated that HNF1A-AS1 significantly enhances U2SURP\u0026apos;s binding to CD44 pre-mRNA (Figure 4H). These findings illustrate that HNF1A-AS1 facilitates the interaction between U2SURP and CD44 pre-mRNA, thereby modulating splicing events.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eU2SURP-induced alternative splicing of CD44 mediates the role of HNF1A-AS1 in facilitating cancer metastasis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe CD44 gene undergoes extensive alternative splicing, generating various isoforms, including the standard isoform CD44s and multiple variant isoforms, CD44v (3-10) (Figure 5A). Gain- and loss-of-function experiments revealed that HNF1A-AS1 upregulates the expression level of CD44v (3-10) while downregulating CD44s (Figure 5B, C).\u0026nbsp;In addition, U2SURP knockdown in PC cells led to the upregulation of CD44s and the downregulation of CD44v (3-10) (Figure 5D).\u003c/p\u003e\n\u003cp\u003eAnalysis of 80 PC tumor tissues alongside matched adjacent normal tissues indicated that CD44s expression was lower and CD44v (3-10) expression was higher in tumors, suggesting that HNF1A-AS1 and U2SURP orchestrate CD44 splicing to favor the production of CD44v (3-10) isoform (Figure 5E). Immunohistochemistry of 31 tumor and adjacent non-tumor tissues further demonstrated a marked increase in CD44v (3-10) expression in PC tissues compared to adjacent normal tissues (Figure 5F). Elevated CD44v (3-10) levels correlated with perineural invasion, distant metastasis, and lymph node metastasis (Table S5). Both TCGA and our dataset revealed that patients with lower CD44v (3-10) expression exhibited longer survival compared to those with higher levels (Figure 5G-H). These findings suggest that HNF1A-AS1-mediated alternative splicing of CD44, shifting from CD44s to CD44v (3-10), may serve as a marker for poor prognosis in patients with PC.\u003c/p\u003e\n\u003cp\u003eTo investigate the functional role of CD44v (3-10), cell lines ectopically expressing CD44s (3-10) or CD44v (3-10) were generated (Figure S6A). Invasion and wound healing assays demonstrated that CD44v (3-10) significantly enhanced the ability of PC cell invasion and metastasis compared to CD44s (Figure S6B-C). Notably, CD44v (3-10) overexpression effectively counteracted the suppression of PC invasion and metastasis induced by HNF1A-AS1 knockdown, both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e (Figure I-K). These results underscore the role of HNF1A-AS1 in interacting with U2SURP to\u0026nbsp;modulate CD44 splicing, thereby promoting cancer metastasis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHNF1A-AS1 was a downstream gene of SNAI2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further elucidate the regulatory networks involving HNF1A-AS1, the interaction between HNF1A-AS1 and SNAI2 was examined. Motif sequences for SNAI2 were sourced from the JASPAR database (Figure 6A), revealing five predicted binding sites on the HNF1A-AS1 promoter (Figure 6B).\u0026nbsp;PCR primers, designed to span approximately 200 bp of these binding and transcription sites, facilitated subsequent analysis (Figure 6C). ChIP-qPCR demonstrated that Flag-tagged SNAI2 successfully precipitated and amplified the HNF1A-AS1 promoter sequence, confirming the direct binding of SNAI2 to this region (Figure 6D). To investigate SNAI2\u0026apos;s effect on transcriptional activity, promoter sequences, including wild-type (Wt) and various mutant binding sites (Mut), were cloned into the pGL4.20 plasmid and transfected into both control and SNAI2-overexpressing PC cells (Figure 6E). Luciferase assays revealed that SNAI2 overexpression enhanced the HNF1A-AS1 promoter activity without altering the SNAI2 binding sites (Figure 6F). This suggests that SNAI2 directly binds to the HNF1A-AS1 promoter to drive its transcription. RT-qPCR further confirmed that SNAI2 overexpression in PC cells elevated HNF1A-AS1 levels, while SNAI2 knockdown reduced its expression (Figure 6G). FISH and immunofluorescence staining of tissue samples also demonstrated co-expression of SNAI2, HNF1A-AS1, and U2SURP in PC tissues (Figure 6H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpecific ASOs suppress tumor progression by controlling HNF1A-AS1 circularization and secretion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe second-generation ASOs designed target HNF1A-AS1, utilizing phosphorothioate backbones and modified oligoribonucleotides to lower intracellular levels of HNF1A-AS1 (Figure 7A). The efficacy of ASO1-HNF1A-AS1 was evaluated both\u0026nbsp;\u003cem\u003ein vitro\u003c/em\u003e and\u0026nbsp;\u003cem\u003ein vivo\u003c/em\u003e. Transwell invasion and wound healing assays demonstrated that ASO1-HNF1A-AS1 markedly inhibited the invasion and migration of PC cells (Figure 7B-C). Western blot analysis confirmed a significant reduction in epithelial-mesenchymal transition (EMT) due to ASO1-HNF1A-AS1 treatment (Figure 7D). Further examination of tumor metastasis in a xenograft model revealed that ASO1-HNF1A-AS1 effectively diminished liver metastasis when PC cells were injected into the mouse spleen, as monitored by IVIS imaging (Figure 7F). Similarly, ASO1-HNF1A-AS1 treatment substantially reduced lung metastases in an experimental lung metastasis model (Figure 7G) and significantly inhibited lymph node metastasis in the footpad inoculation model (Figure 7H). No notable changes in organ morphology or levels of key blood parameters\u0026mdash;such as monocytes, white blood cells, lymphocytes, granulocytes, red blood cells, hemoglobin, platelets, hematocrit, aspartate aminotransferase, and alanine aminotransferase\u0026mdash;were observed in ASO1-HNF1A-AS1-treated mice, indicating the absence of toxic effects (Figure S7). Collectively, these results suggest that ASO1-HNF1A-AS1 holds promise as a novel therapeutic approach for cancer treatment.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn recent years, lncRNAs have emerged as pivotal players in various aspects of cancer biology, including tumor metabolism, proliferation, metastasis, and chemotherapy resistance, garnering significant attention in cancer research [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. To evaluate the role of HNF1A-AS1 in PC, its expression levels were measured using qRT-PCR and in situ hybridization (ISH) across bile duct adjacent/primary tissues, normal/cancer cell lines, and metastatic tumor samples. Prognostic implications were further explored through survival analysis. Both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e studies revealed that HNF1A-AS1 knockdown substantially impaired cell migration and invasion, suggesting that HNF1A-AS1 may function as an oncogene in PC and is critical to disease progression. This study also investigated the mechanism underlying the upregulation of HNF1A-AS1 in PC cells. It was found that SNAI2 specifically binds to the promoter region of HNF1A-AS1, elucidating how SNAI2 contributes to its expression.\u003c/p\u003e \u003cp\u003eProteomic analyses, including RNA-seq, RNA pull-down assays, and mass spectrometry, identified U2 snRNP-associated SURP motif-containing protein (U2SURP) as a key interactor with HNF1A-AS1. U2SURP, a serine/arginine-rich protein and a component of the 17S U2 snRNP complex, features a highly phosphorylated SR domain at the carboxy terminus and an RNA recognition motif (RRM) at the amino terminus [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Despite its importance, the regulatory mechanisms and biological functions of U2SURP remain underexplored. Previous research by De Maio et al. has shown that U2SURP regulates splicing and gene expression by interacting with other splicing factors, such as RBM17 and CHEP [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. AS is a vital process that enhances gene expression and transcriptome diversity in eukaryotic cells, enabling a single precursor mRNA to generate multiple mRNA and protein variants [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Over 95% of human gene transcripts with multiple exons undergo AS [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], with different splice variants being expressed across various tissues and cells [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Dysregulation of AS can contribute to a range of human diseases. This study demonstrates that the lncRNA HNF1A-AS1 and U2SURP are involved in CD44 alternative splicing, leading to EMT activation.\u003c/p\u003e \u003cp\u003eThe spliceosomal variants of CD44 exhibit different roles across various tumors [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Some studies suggest that the transition from CD44v to CD44s can facilitate tumor progression [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], while others indicate that CD44v promotes tumor development [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Conversely, the conversion from CD44s to CD44v has been associated with accelerated tumor progression [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. This variability in function highlights the controversy surrounding the roles of different CD44 splice isoforms in PC [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Our research identified CD44v (3\u0026ndash;10) as a key player in tumor progression. Knockdown of HNF1A-AS1 significantly reduced CD44 precursor mRNA splicing to CD44v (3\u0026ndash;10), thereby inhibiting tumor advancement. Overexpression of CD44v (3\u0026ndash;10) in PC cells further confirmed its role in promoting tumor progression, suggesting that targeting the alternative splicing of CD44v (3\u0026ndash;10) could offer a novel therapeutic strategy.\u003c/p\u003e \u003cp\u003eMetastatic cancer accounts for approximately 90% of cancer-related deaths worldwide, yet the mechanisms underlying cancer metastasis remain poorly understood [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Current treatments for metastatic cancer are inadequate. Our study reveals that HNF1A-AS1 enhances the alternative splicing of CD44 pre-mRNA by interacting with the splicing factor U2SURP, leading to increased production of CD44v (3\u0026ndash;10). This splice variant, a novel regulator of cancer metastasis, promotes tumor invasion and dissemination, positioning it as a potential therapeutic target. Two decades ago, the ability of ASOs to influence RNA processing and protein expression was first discovered. Recent clinical trials have demonstrated the effectiveness of ASO-based therapies [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Our study shows that ASOs targeting HNF1A-AS1 significantly inhibit tumor invasion and metastasis in both cellular and animal models.\u003c/p\u003e \u003cp\u003eIn conclusion, this study provides the first evidence that SNAI2 specifically binds to the HNF1A-AS1 promoter and enhances its expression in PC. The interaction between HNF1A-AS1 and U2SURP facilitates the alternative splicing of CD44, shifting from the standard isoform CD44s to the variant CD44v (3\u0026ndash;10), which promotes cancer invasion and metastasis. This underscores the critical role of CD44 splicing in cancer progression and suggests that ASOs targeting HNF1A-AS1 could be a promising approach for treating PC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no potential conflicting interests associated with their research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTengxiang Chen designed the experiments and drafted the manuscript; Shan Lei, Zhirui Zeng, Zhixue Zhang, Yating Sun, Wenpeng Cao, Jigang Pan, Tuo Zhang, Yingmin Wu and Dahuan Li conducted and processed the data. All authors reviewed the final version of the manuscript and granted approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Human Research Ethics Review Committee of Guizhou Medical University approved the application of these clinical samples, which was performed according to the tenets of the Declaration of Helsinki. The animal experiments were approved by Animal Ethics Committee of Guizhou Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the\u0026nbsp;National Natural Science Foundation of China (82103681), the Guizhou Medical University National Natural Science Foundation Cultivation Project (grant numbers 20NSP020 and 19NSP034), the Guizhou Provincial Science and Technology Projects (No. ZK [2024]159) and Postgraduate Research Fund of Guizhou Province (No. YJSKYJJ [2021] 143), the Continuous Support Fund for Excellent Scientific Research Platform of Colleges and Universities in Guizhou Province (QJJ (2022) 020), Discipline Leading Talents Project of the Affiliated Hospital of Guizhou Medical University (No.gyfyxkyc-2023-01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eKlein AP. Pancreatic cancer epidemiology: understanding the role of lifestyle and inherited risk factors. Nat Rev Gastroenterol Hepatol. 2021;18(7):493-502.\u003c/li\u003e\n \u003cli\u003ePark W, Chawla A, O\u0026apos;Reilly EM. Pancreatic Cancer: A Review. JAMA. 2021; 326(9):851-862.\u003c/li\u003e\n \u003cli\u003eCollisson EA, Bailey P, Chang DK, Biankin AV. Molecular subtypes of pancreatic cancer. Nat Rev Gastroenterol Hepatol. 2019; 16(4):207-220.\u003c/li\u003e\n \u003cli\u003eRansohoff JD, Wei Y, Khavari PA. The functions and unique features of long intergenic non-coding RNA. Nat Rev Mol Cell Biol. 2018; 19(3):143-157.\u003c/li\u003e\n \u003cli\u003eKopp F, Mendell JT. Functional Classification and Experimental Dissection of Long Noncoding RNAs. Cell. 2018; 172(3):393-407.\u003c/li\u003e\n \u003cli\u003eRansohoff JD, Wei Y, Khavari PA. The functions and unique features of long intergenic non-coding RNA. Nat Rev Mol Cell Biol. 2018; 19(3):143-157.\u003c/li\u003e\n \u003cli\u003eBradley RK, Anczuk\u0026oacute;w O. RNA splicing dysregulation and the hallmarks of cancer. Nat Rev Cancer. 2023; 23(3):135-155.\u003c/li\u003e\n \u003cli\u003eZhang Y, Qian J, Gu C, Yang Y. Alternative splicing and cancer: a systematic review. Signal Transduct Target Ther. 2021;6(1):78.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBaralle FE, Giudice J. Alternative splicing as a regulator of development and tissue identity. Nat Rev Mol Cell Biol. 2017; 18(7):437-451.\u003c/li\u003e\n \u003cli\u003eNaro C, Antonioni A, Medici V, Caggiano C, Jolly A, de la Grange P, et al.\u0026nbsp; Splicing targeting drugs highlight intron retention as an actionable vulnerability in advanced prostate cancer. J Exp Clin Cancer Res. 2024; 43(1):58.\u003c/li\u003e\n \u003cli\u003eUle J, Blencowe BJ. Alternative Splicing Regulatory Networks: Functions, Mechanisms, and Evolution. Mol Cell. 2019;76(2):329-345.\u003c/li\u003e\n \u003cli\u003eSciarrillo R, Wojtuszkiewicz A, Assaraf YG, Jansen G, Kaspers GJL, Giovannetti E, et al.\u0026nbsp;The role of alternative splicing in cancer: From oncogenesis to drug resistance. Drug Resist Updat. 2020; 53:100728.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eChan YT, Lu Y, Wu J, Zhang C, Tan HY, Bian ZX, et al.\u0026nbsp;CRISPR-Cas9 library screening approach for anti-cancer drug discovery: overview and perspectives. Theranostics. 2022 Apr 11;12(7):3329-3344.\u003c/li\u003e\n \u003cli\u003eXu WW, Liao L, Dai W, Zheng CC, Tan XP, He Y, et al. Genome-wide CRISPR/Cas9 screening identifies a targetable MEST-PURA interaction in cancer metastasis. EBioMedicine. 2023 Jun;92: 104587. \u0026nbsp;\u003c/li\u003e\n \u003cli\u003eJoung J, Engreitz JM, Konermann S, Abudayyeh OO, Verdine VK, Aguet F, et al. Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood. Nature. 2017 Aug 17;548(7667):343-346.\u003c/li\u003e\n \u003cli\u003eHassn Mesrati M, Syafruddin SE, Mohtar MA, Syahir A. CD44: A Multifunctional Mediator of Cancer Progression. Biomolecules. 2021; 11(12):1850.\u003c/li\u003e\n \u003cli\u003eYan Y, Zuo X, Wei D. Concise Review: Emerging Role of CD44 in Cancer Stem Cells: A Promising Biomarker and Therapeutic Target. Stem Cells Transl Med. 2015; 4(9):1033-43.\u003c/li\u003e\n \u003cli\u003eLuo H, Li J, Lin Q, Xiao X, Shi Y, Ye X, et al. \u0026nbsp;Ultrasonic irradiation and SonoVue microbubbles-mediated RNA interference targeting PRR11 inhibits breast cancer cells proliferation and metastasis, but promotes apoptosis. Biosci Rep. 2020 ;40(11): BSR20201854.\u003c/li\u003e\n \u003cli\u003eJoung J, Engreitz JM, Konermann S, Abudayyeh OO, Verdine VK, Aguet F, et al. Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood. Nature. 2017 Aug 17;548(7667):343-346.\u003c/li\u003e\n \u003cli\u003eZeng Z, Lei S, Wang J, Yang Y, Lan J, Tian Q, et al. \u0026nbsp;A novel hypoxia-driven gene signature that can predict the prognosis of hepatocellular carcinoma. Bioengineered. 2022;13(5):12193-12210.\u003c/li\u003e\n \u003cli\u003eFeng Z, Li K, Qin K, Liang J, Shi M, Ma Y,\u0026nbsp;et al.\u0026nbsp; The LINC00623/NAT10 signaling axis promotes pancreatic cancer progression by remodeling ac4C modification of mRNA. J Hematol Oncol. 2022;15(1):112.\u003c/li\u003e\n \u003cli\u003eJin B, Zhu J, Pan T, Yang Y, Liang L, Zhou Y, et al.\u0026nbsp;MEN1 is a regulator of alternative splicing and prevents R-loop-induced genome instability through suppression of RNA polymerase II elongation. Nucleic Acids Res. 2023;51(15):7951-7971.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eTaniue K, Akimitsu N. The Functions and Unique Features of LncRNAs in Cancer Development and Tumorigenesis. Int J Mol Sci. 2021;22(2):632.\u003c/li\u003e\n \u003cli\u003eEptaminitaki GC, Stellas D, Bonavida B, Baritaki S. Long non-coding RNAs (lncRNAs) signaling in cancer chemoresistance: From prediction to druggability. Drug Resist Updat. 2022; 65:100866.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDeng L, Liao L, Zhang YL, Hu SY, Yang SY, Ma XY, et al. MYC-driven U2SURP regulates alternative splicing of SAT1 to promote triple-negative breast cancer progression. Cancer Lett. 2023; 560:216124.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eNameki N, Takizawa M, Suzuki T, Tani S, Kobayashi N, Sakamoto T, et al. \u0026nbsp;Structural basis for the interaction between the first SURP domain of the SF3A1 subunit in U2 snRNP and the human splicing factor SF1. Protein Sci. 2022;31(10): e4437.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDe Maio A, Yalamanchili HK, Adamski CJ, Gennarino VA, Liu Z, Qin J, et al.\u0026nbsp;RBM17 Interacts with U2SURP and CHERP to Regulate Expression and Splicing of RNA-Processing Proteins. Cell Rep. 2018;25(3):726-736.e7.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMarasco LE, Kornblihtt AR. The physiology of alternative splicing. Nat Rev Mol Cell Biol. 2023;24(4):242-254.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLiu Y, Liu X, Lin C, Jia X, Zhu H, Song J, et al. \u0026nbsp;Noncoding RNAs regulate alternative splicing in Cancer. J Exp Clin Cancer Res. 2021;40(1):11.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eArtemaki PI, Kontos CK. Alternative Splicing in Human Physiology and Disease. Genes (Basel). 2022;13(10):1820.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eTao Y, Zhang Q, Wang H, Yang X, Mu H. Alternative splicing and related RNA binding proteins in human health and disease. Signal Transduct Target Ther. 2024;9(1):26.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eZhang H, Brown RL, Wei Y, Zhao P, Liu S, Liu X, et al. CD44 splice isoform switching determines breast cancer stem cell state. Genes Dev. 2019;33(3-4):166-179.\u003c/li\u003e\n \u003cli\u003eBhattacharya R, Mitra T, Ray Chaudhuri S, Roy SS. Mesenchymal splice isoform of CD44 (CD44s) promotes EMT/invasion and imparts stem-like properties to ovarian cancer cells. J Cell Biochem. 2018;119(4):3373-3383.\u003c/li\u003e\n \u003cli\u003eGuo L, Ke H, Zhang H, Zou L, Yang Q, Lu X, et al. TDP43 promotes stemness of breast cancer stem cells through CD44 variant splicing isoforms. Cell Death Dis. 2022;13(5):428.\u003c/li\u003e\n \u003cli\u003eGiraud J, Seeneevassen L, Rousseau B, Bouriez D, Sifr\u0026eacute; E, Giese A, et al.\u0026nbsp;CD44v3 is a marker of invasive cancer stem cells driving metastasis in gastric carcinoma. Gastric Cancer. 2023;26(2):234-249.\u003c/li\u003e\n \u003cli\u003eZhu H, Zhou W, Wan Y, Lu J, Ge K, Jia C. CD44V3, an Alternatively Spliced Form of CD44, Promotes Pancreatic Cancer Progression. Int J Mol Sci. 2022;23(20):12061.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLiu YN, Tsai MF, Wu SG, Chang TH, Shih JY. CD44s and CD44v8-10 isoforms confer acquired resistance to osimertinib by activating the ErbB3/STAT3 signaling pathway. Life Sci. 2024; 336:122345.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eChew SH, Okazaki Y, Akatsuka S, Wang S, Jiang L, Ohara Y, et al. Rheostatic CD44 isoform expression and its association with oxidative stress in human malignant mesothelioma. Free Radic Biol Med. 2017; 106:91-99.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLi Z, Chen K, Jiang P, Zhang X, Li X, Li Z. CD44v/CD44s expression patterns are associated with the survival of pancreatic carcinoma patients. Diagn Pathol. 2014;9:79.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eGerstberger S, Jiang Q, Ganesh K. Metastasis. Cell. 2023;186(8):1564-1579.\u003c/li\u003e\n \u003cli\u003eFares J, Fares MY, Khachfe HH, Salhab HA, Fares Y. Molecular principles of metastasis: a hallmark of cancer revisited. Signal Transduct Target Ther. 2020;5(1):28.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMa WK, Voss DM, Scharner J, Costa ASH, Lin KT, Jeon HY, et al. ASO-Based PKM Splice-Switching Therapy Inhibits Hepatocellular Carcinoma Growth. Cancer Res. 2022;82(5):900-915.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eRamasamy T, Ruttala HB, Munusamy S, Chakraborty N, Kim JO. Nano drug delivery systems for antisense oligonucleotides (ASO) therapeutics. J Control Release. 2022; 352:861-878.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHe K, Barsoumian HB, Puebla-Osorio N, Hu Y, Sezen D, Wasley MD, et al. Inhibition of STAT6 with Antisense Oligonucleotides Enhances the Systemic Antitumor Effects of Radiotherapy and Anti-PD-1 in Metastatic Non-Small Cell Lung Cancer. Cancer Immunol Res. 2023;11(4):486-500.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"pancreatic cancer, HNF1A-AS1, alternative splicing, U2SURP, CD44, antisense oligonucleotides","lastPublishedDoi":"10.21203/rs.3.rs-5388928/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5388928/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eLong non-coding RNAs (LncRNAs) have emerged as pivotal biomarkers and regulators across various cancers. In pancreatic cancer (PC), however, the mechanisms underlying the expression and functional roles of lncRNAs remain inadequately elucidated.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eCRISPR/CRISPR-associated protein 9 (Cas9) single-guide RNA (sgRNA)-pooled lncRNA libraries were used to screen for the critical lncRNAs regulating PC metastasis. The expression levels of lncRNA HNF1A-AS1 were quantified in PC cell lines and clinical samples using qRT-PCR. Investigations into HNF1A-AS1's impact on PC cell migration and invasion were conducted through both loss-of-function and gain-of-function approaches. A range of techniques, including fluorescence in situ hybridization (FISH), mRNA sequencing, RNA immunoprecipitation (RIP), bioinformatics analysis, dual-luciferase reporter assays, RNA pull-down assays, ChIP-PCR, and rescue experiments, were employed to unravel the competitive endogenous RNA (ceRNA) network regulated by HNF1A-AS1.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe research identified HNF1A-AS1 as a novel and influential lncRNA that acts as a pro-metastatic factor in PC. Compared to normal controls, HNF1A-AS1 levels were significantly elevated in PC cell lines and tissue samples. Elevated HNF1A-AS1 expression correlated with increased lymph node metastasis and poorer overall survival in patients with PC. Knocking down HNF1A-AS1 substantially reduced metastasis, whereas its overexpression exacerbated it. Mechanistically, HNF1A-AS1 promotes an oncogenic splice switch from the standard isoform CD44s to the variant isoform CD44v (3\u0026ndash;10), acting as a scaffold for the binding of CD44 pre-mRNA to U2SURP. The levels of HNF1A-AS1 and CD44v (3\u0026ndash;10) serve as indicators of poor prognosis. Furthermore, SNAI2 was shown to specifically bind to the HNF1A-AS1 promoter, thereby activating its transcription. Antisense oligonucleotides (ASOs) targeting HNF1A-AS1 also significantly inhibited cancer metastasis.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eSNAI2\u0026rsquo;s role in enhancing HNF1A-AS1 transcription underscores the critical function of HNF1A-AS1 in promoting PC metastasis through modulation of CD44 alternative splicing \u003cem\u003evia\u003c/em\u003e U2SURP. Targeted silencing of HNF1A-AS1 presents a promising therapeutic avenue for patients with PC.\u003c/p\u003e","manuscriptTitle":"Genome-scale activation screen reveals lncRNA HNF1A-AS1 as a novel therapeutic target for pancreatic cancer metastasis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-24 17:07:18","doi":"10.21203/rs.3.rs-5388928/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6737152e-ad99-4ea4-84f9-9b8655015027","owner":[],"postedDate":"December 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":41713907,"name":"Biological sciences/Cell biology/Cell migration/Cell invasion"},{"id":41713908,"name":"Biological sciences/Molecular biology/Non-coding RNAs/Long non-coding RNAs"},{"id":41713909,"name":"Biological sciences/Cancer/Cancer therapy/Drug development"}],"tags":[],"updatedAt":"2025-01-03T12:40:58+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-24 17:07:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5388928","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5388928","identity":"rs-5388928","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}