Requirement of splicing factor hnRNP A2B1 of hnRNPs for tumorigenesis of melanoma stem cells

Requirement of splicing factor hnRNP A2B1 of hnRNPs for tumorigenesis of melanoma stem cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Requirement of splicing factor hnRNP A2B1 of hnRNPs for tumorigenesis of melanoma stem cells Mengqi Chu, Haitao Wan, Xiaobo Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-92870/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Jan, 2021 Read the published version in Stem Cell Research & Therapy → Version 1 posted 13 You are reading this latest preprint version Abstract Background: Cancer stem cells play essential roles in tumorigenesis, thus being the important targets for tumor therapy. The hnRNP family proteins, the important splicing factors, are found to be associated with tumor progression. However, the influence of hnRNPs on cancer stem cells has not been extensively explored. Methods: Quantitative real-time PCR and Western blot were used to examine the gene expression level. RNA immunoprecipitation assay and RNA sequencing were conducted to identify the RNAs interacted with hnRNP A2B1 on a genome-wide scale. The in vivo assays were performed in nude mice. Results: In this study, the results showed that hnRNP A2B1 of 19 hnRNPs was significantly upregulated in melanoma stem cells compared with non-stem cells, suggesting the important role of hnRNP A2B1 in cancer stem cells. The hnRNP A2B1 silencing triggered the cell cycle arrest in G2 phase, leading to apoptosis of melanoma stem cells. The results revealed that hnRNP A2B1 could bind to the precursor mRNAs of pro-apoptosis genes ( DAPK1, SYT7 and RNF128 ) and anti-apoptosis genes ( EIF3H, TPPP3 and DOCK2 ) to regulate the splicing of these 6 genes, thus promoting the expressions of anti-apoptosis genes and suppressing the expressions of pro-apoptosis genes. The in vivo data indicated that hnRNP A2B1 was required for tumorigenesis of melanoma stem cells in vivo by affecting the splicing of TPPP3 , DOCK2 , EIF3H , RNF128 , DAPK1 and SYT7 , thus suppressing apoptosis of melanoma stem cells. Conclusions: HnRNP A2B1 was required for tumorigenesis of melanoma stem cells. Therefore our findings presented novel molecular insights into the roles of hnRNPs in cancer stem cells. Stem Cell & Developmental Cell Biology Melanoma cancer stem cells hnRNP apoptosis tumorigenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Cancer is known as a major cause of death due to its high morbidity and mortality [ 1 ]. A lot of investigations have suggested that only a small proportion of cancer cells have tumorigenic capability, which is named as cancer stem cells (CSCs) [ 2 ]. CSCs are proved to play essential roles in predicting the biological aggressiveness of cancers. They are correlated with recurrence, metastasis and poor survival in solid tumors [ 3 ], thus being responsible for tumorigenesis, tumor differentiation, tumor maintenance, tumor spread, and tumor relapse. CSCs have the ability of unlimited growth and self-renewal [ 4 ]. Therefore, elimination of CSCs is difficult but essential to cure cancer completely [ 5 ]. At present, it is believed that CSCs origin from abnormal expressions of a series of genes. Gene expression regulation plays an essential role in the biogenesis of CSCs. The regulation of gene expression depends on many factors, including DNA sequence, transcriptional regulation, post-transcriptional modification, translational control and protein modification [ 6 , 7 ]. Post-transcriptional regulation is the most important regulation process, because it can influence RNA stability, localization, translation efficiency and sequence [ 8 ]. It also contributes to shaping tissue-type specific proteomes [ 9 ]. Post-transcriptional regulation includes transcript stability, binding of RNAs to RNA-binding proteins (RBPs), alternative splicing and regulations by microRNAs (miRNAs) [ 10 ], thus providing the flexibility for cells to adapt to a wide range of physiological conditions. Among post-transcriptional regulations, alternative splicing is one of the crucial mechanisms for gene expression [ 11 ]. It is well known that alternative splicing often results in differential intron and exon retention or skipping and this process can produce different mRNA and protein isoforms from one gene. Studies have shown that over 90% of human RNAs are alternatively spliced, leading to a cellular mRNA accumulation that can encode four to five fold more proteins than protein-coding genes in the genome [ 12 ]. Alternative splicing occurs in a large ribonucleoprotein (RNP) machine called spliceosome [ 13 ]. The spliceosome contains five different small nuclear RNP (snRNP) subunits (U1, U2, U4, U5 and U6) along with more than 200 associated protein cofactors [ 14 ]. The splicing process involves 70–250 splicing factors depending on the species, which can interact in multiple steps with RNA and proteins [ 15 ]. Among splicing factors, serine/arginine (SR) proteins and heterogenous ribonuclear proteins (hnRNPs) are the crucial families of splicing factors [ 16 , 17 ]. At present, 12 classical SR proteins and 17 canonical hnRNPs are identified [ 18 ]. Most SR proteins act as splicing activators by binding precursor mRNAs (pre-mRNAs) at exonic splicing enhancers. SR proteins often compete with splicing repressors, such as hnRNPs, whose binding to exonic or intronic splicing silencers can inhibit the selection of splicing site, thereby modulating alternative splicing [ 19 ]. By binding to different splicing factors, pre-mRNAs are able to be alternatively spliced in different ways [ 20 ]. As reported, some hnRNPs, such as hnRNP A1, K and A2B1, are aberrantly higher expressed in lung cancer, breast cancer or hepatocellular carcinoma [ 21 – 23 ], suggesting the important roles of hnRNPs in tumorigenesis. However, the influence of hnRNPs on cancer stem cells has not been extensively explored. To address this issue, the expression levels of hnRNPs were examined in melanoma stem cells. The results showed that hnRNP A2B1 of 19 hnRNPs was significantly upregulated in melanoma stem cell compared with melanoma non-stem cells. The further investigations revealed that hnRNP A2B1 took great effects on tumorigenesis of melanoma stem cells. Methods Cell culture Melanoma stem cells and non-stem cells were sorted in our laboratory previously from cell line MDA-MB-435 [24] or A375 [25]. Melanoma stem cells and non-stem cells were cultured in DMEM/F-12 medium (Invitrogen, USA) supplemented with 20 ng/mL epidermal growth factor (EGF) (Beyotime Biotechnology, China), 10 ng/mL basic fibroblast growth factor (bFGF) (Beyotime Biotechnology), 5 mg/mL of insulin (Beyotime Biotechnology), and 2% of B-27 (Sigma, USA) at 37℃ in a humidified atmosphere with 5% CO 2 . Quantification of mRNA with real-time PCR Total RNAs were extracted from cells using an RNA Isolation Kit (Ambion, USA). The reverse transcription reaction was conducted with PrimeScript RT Reagent Kit (TaKaRa, Japan). Quantitative real-time PCR was performed using 2×ChamQ SYBR qPCR Master Mix (Vazyme, USA). The PCR reaction mixture (10 μL) contained Rox reference Dye, cDNA, ChamQ SYBR qPCR Master Mix (Vazyme) and primers (Table S1). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was included for normalization. The 2 -( △△ Ct) method was used to calculate the relative fold change of mRNA expression [26]. PCR was conducted by maintaining the reaction at 95℃ for 30 s, and then alternating for 40 cycles between 95℃ for 5 s and 60℃ for 30 s. Western blot analysis Proteins (about 50 μg/protein) were separated using 12% SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with triethanolamine-buffered saline solution (TBS) containing 5% skim milk. Subsequently, the membrane was incubated overnight with a primary antibody, followed by incubation with the alkaline phosphatase-conjugated secondary antibody (Roche, Switzerland) for 2 h at room temperature. After rinses, the membrane was detected with BCIP/NBT substrate (Sangon Biotech, China). All antibodies were purchased from Proteintech Group (USA) at catalog number 67445-1-Ig (hnRNP A2B1), 15057-1-AP (TPPP3), 11310-1-AP (EIF3H), 25136-1-AP (DAPK1), A12757 (SYT7), 66969-1-Ig (DOCK2) and 26015-1-AP (RNF128). Northern blot Total RNAs were extracted using a cell/tissue genomic DNA extraction kit (Generay Biotech, China) according to the manufacturer’s manual. After electrophoresis of 30 μg RNAs on a 2% agarose gel, the RNAs were transferred to a nylon membrane (Amersham Biosciences, Sweden) for 1 h, followed by ultraviolet cross-linking. The membrane was prehybridized in prehybridization solution (Roche, Switzerland) for 30 min. Subsequently, the membrane was hybridized with a DIG-labeled probe (Table S2) at 42 0 C overnight. The membrane was rinsed and then blocked in a blocking solution (Roche) for 1 h at room temperature. After incubation with the antibody (10 μl) against DIG-labeled alkaline phosphatase (Roche) for 2 h at room temperature, the signals of the membrane were detected with the substrate BCIP/NBT solution (Roche). RNA immunoprecipitation (RIP) assay Cells were treated with paraformaldehyde for 10 min at room temperature. After washes with ice-cold phosphate buffer saline (PBS), the cells were incubated with PBS containing 0.125 M glycine for 5 min and then with hypotonic buffer [10 mM N-2-hydroxyethylpiperazine-N-ethane-sulphonicacid (HEPES), 1.5 mM MgCl 2 , 10 mM KCl, 0.4% Nonidet P-40, pH 7.9] on ice for 15 min, followed by centrifugation at 3,000 ×g for 7 min. The pellet was resuspended in sonication buffer [10% sodium dodecyl sulfate (SDS), 0.5 M ethylene diamine tetraacetic acid (EDTA), 1 M Tris-HCl, pH 8.0] and then subjected to ultrasonication. The sample was centrifuged at 12,000 ×g for 20 s and the supernatant was incubated with antibody-coupled Protein G magnetic beads (70μl) (Bio-Rad Laboratories, USA) at 4℃ overnight. The beads were washed with PBS. Subsequently the RNAs were extracted using RNA Isolation Kit (Ambion, USA). RNA-seq and data analysis The extracted RNAs were subjected to RNA-seq using an Illumina Hiseq 2500 system by Novogene Corporation (China). Briefly, the rRNAs were removed by ribo-zerotm kit (Epicentre, France). Subsequently fragmentation buffer was added to break the RNA into short segments of 250-300bp, followed by the synthesis of cDNAs with random hexamers. After purification with AMPure XP beads, the double-stranded cDNAs were added with A tails and the connection of sequencing joints. The cDNA library was enriched by PCR. Sequencing was performed. After assembly of RNA-seq data, the raw data was processed to remove the sequences of adapter, ploy-N and low-quality reads. The clean reads were aligned to the genome reference consortium human reference 38 (hg38) by BWA (Burrows Wheeler Aligner) and IGV (Integrative Genomics Viewer). Based on the counts of reads, the gene expression profile was obtained. Kyoto encyclopedia of genes and genomes (KEGG) analysis The coding sequences of transcripts were extracted and used as queries to search the protein sequences collected in the GO (gene ontology) database with the blast E-value of less than 1×10 -5 . The best hit GO identities were assigned to the transcripts. The p-values were corrected for false discovery rate. Deduced genes with homologues in other organisms were used to map to conserved biological pathways. Semi-quantitative reverse transcription (RT)-PCR Total RNAs were extracted from cells using a cell/tissue genomic DNA extraction kit (Generay Biotech, China) and then quantified by NanoDrop ND-1000 spectrophotometer. The complementary DNA was synthesized using HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, USA) following the manufacturer’s instructions. Subsequently PCR was conducted with sequence-specific primers (Table S3). β-tubulin was used as a loading control. Cell viability analysis Cell viability was evaluated using MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (Promega, USA). Cells were seeded onto a 96-well plate. Thirty-six hours later, the plate was incubated with 20 μL of MTS reagent for 1 h at 37℃. Subsequently the absorbance was recorded at 490 nm. All experiments were repeated three times. Analysis of caspase 3/7 activity Caspase-Glo 3/7 assay (Promega) was used to evaluate the activity of caspase 3/7 according to the manufacturer’s protocol. Cells at a density of 1×10 4 /well were plated onto a 96-well plate. Subsequently 50 mL of caspase-Glo 3/7 reagent (Promega) was added to each well. After incubation in the dark at room temperature for 1 h, the luminescence of cells was measured. Cell cycle analysis Cell cycle analysis was conducted with flow cytometry. Cells were fixed in ice-cold ethanol overnight. Then the cells were incubated with DNase-free RNase A (20 mg/mL) for 30 min. After centrifugation at 500×g for 5 min, the cells were stained with propidium iodide (PI, 50 mg/mL). The fluorescence intensity of 1×10 4 cells was measured with a flow cytometer at an excitation wavelength of 488 nm. Silencing and overexpression of gene in cells To silence the gene expression in cancer stem cells, RNA interference (RNAi) assay was conducted using gene-specific siRNA (Table S4). The melanoma stem cells (1×10 5 ) were transfected with 50 nM of siRNA using Lipofectamine 2000 (Invitrogen, USA). All the siRNAs were synthesized by Shanghai GenePharma Co., Ltd. At different time after transfection, the cells were harvested for later use. To overexpress a gene in cancer stem cells, the gene was amplified using PCR with sequence-specific primers (Table S5), followed by cloning into pcDNA3.1 (+) vector. The recombinant plasmid was transfected into melanoma stem cells using Lipofectamine 2000 (Invitrogen). At different time after transfection, the cells were collected for later use. To overexpress hnRNP A2B1 in the hnRNP A2B1-silenced melanoma stem cells, hnRNP A2B1 was mutated at a nucleotide (position 32 A→T) to prevent the recognition by hnRNP A2B1-siRNA. Briefly hnRNP A2B1 was amplified by hnRNP A2B1 primers 1 and 4 (Table S5), and hnRNP A2B1 primers 2 and 3 (Table S5), respectively. Then the amplified products were subjected to PCR with hnRNP A2B1 primers 1 and 2 (Table S5), followed by cloning into pcDNA3.1 (+) vector. Tumorigenicity in nude mice Melanoma stem cells were transfected with hnRNP A2B1-shRNA (short hairpin RNA) (5’-AGGAACAGTTCCGTAAGCTCTTTAT-3’) to stably silence the expression of hnRNP A2B1. ShRNA was designed with invitrogen BLOCK-iT™ RNAi Designer (http://rnaidesigner.thermofisher.com). As a control, the sequence of hnRNP A2B1-shRNA was randomly scrambled, generating hnRNP A2B1-shRNA-scrambled (5’-CCGGGCGCGATAGCGCTAATAGCGA-3’). ShRNA was cloned into lentiviral vector pLent-U6-GFP-Puro (Vigene Bioscience, USA), followed by transfection into 293T cells using Lipofectamine 2000 reagent (Life Technologies, USA). At 48 h after transfection, the viral particles were collected to infect melanoma stem cells. Subsequently, the cells were cultured in medium contained 10 µg/ml puromycin for three days. After puromycin screening, only the cells expressing green fluorescence protein (GFP) were selected as stable strains expressing shRNA. Melanoma stem cells transfected with hnRNP A2B1-shRNA or hnRNP A2B1-shRNA-scrambled were suspended in physiological saline. Matrigel (Becton, Dickinson and Company, USA) was added to the cell suspension at the final concentration of 33%. Subsequently, 100 μL of the cell suspension was subcutaneously injected into each BALB/c mouse to induce tumor growth. The tumor volume was measured every week. Six weeks later, the mice were sacrificed. The tumor sizes and tumor weights were determined. All procedures conducted on mice in this study were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC). All the methods were carried out in accordance with the approved guidelines. Immunohistochemical analysis Tumor tissue was cut into 5 μm pieces and mounted onto a slide. The slide was dewaxed and hydrated in 100%, 95% and 80% ethanol for 5 min, respectively. Subsequently the primary antibody was incubated with the slide. After washing with PBS, the slide was incubated with the secondary antibody for 10 min at room temperature. Streptavidin peroxidase was added to the slide, followed by incubation for 10 min at room temperature. Then 3-amino-9-ethylcarbazole (AEC) buffer and AEC chromogen (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were mixed and added to the slide. The slide was incubated for 10 min at room temperature. The proteins and the nucleus were labeled with diaminobenzidine (DAB) (Sigma, USA) or 4’,6-diamidino-2- phenylindole (DAPI), respectively. Statistical analysis The numerical data were analyzed by one-way analysis of variance (ANOVA). The differences between different treatments were analyzed by Student’s t test. All data were presented as mean ± standard deviation. Results Differentially expressed hnRNPs in melanoma stem cells and non-stem cells To explore the roles of hnRNPs in cancer stem cells, the expression levels of 19 hnRNP family genes were characterized in two types of melanoma stem cells and non-stem cells. The melanoma stem cells were previously sorted in our laboratory from melanoma cell lines MDA-MB-435 and A375 [ 24 , 25 ], respectively. The results of quantitative real-time PCR revealed that compared with the controls, 3 hnRNPs (A2B1, I and L) and 8 hnRNPs (A2B1, C, E1, H1, H2, I, K and L) were significantly upregulated in MDA-MB-435 stem cells and in A375 stem cells compared with melanoma non-stem cells (Fig. 1 A), respectively. These data indicated that hnRNP A2B1, hnRNP I and hnRNP L were highly expressed in two types of melanoma cancer stem cells. As shown in Fig. 1 B, the expression profiles of hnRNP I and hnRNP L proteins were similar in MDA-MB-435 melanoma stem cells and non-stem cells, while the hnRNP A2B1 protein was significantly upregulated in melanoma stem cells compared with non-stem cells (Fig. 1 B). Western blot revealed that the content of hnRNP A2B1 was much higher in A375 melanoma stem cells than that in non-stem cells (Fig. 1 C). Therefore, hnRNP A2B1 was further characterized in melanoma stem cells. Requirement of hnRNP A2B1 for melanoma stem cells To investigate the role of hnRNP A2B1 in melanoma stem cells, the hnRNP A2B1 expression was knocked down or rescued, followed by the evaluation of stem cell viability. Northern blots showed that the hnRNP A2B1 mRNA was knocked down in melanoma stem cells by hnRNP A2B1-siRNA compared with the control (Fig. 2 A). To confirm the efficiency of hnRNP A2B1-siRNA, another siRNA specifically targeting hnRNP A2B1 (hnRNP A2B1-siRNA-2) was transfected into melanoma stem cells. The results indicated that the efficiency of hnRNP A2B1-siRNA-2 was similar to that of hnRNP A2B1-siRNA (Fig. 2 A). To rescue the expression of hnRNP A2B1 in hnRNP A2B1-silenced melanoma stem cells, the stem cells were co-transfected with hnRNP A2B1-siRNA and the plasmid expressing hnRNP A2B1. The data of Northern blot analysis revealed that the expression of hnRNP A2B1 was rescued in the hnRNP A2B1-siRNA-treated melanoma stem cells (Fig. 2 A). Western blot yielded essentially the similar results (Fig. 2 B). These data indicated that hnRNP A2B1 was silenced in melanoma stem cells or rescued in the hnRNP A2B1-silenced melanoma stem cells. The results of MTS assays indicated that the hnRNP A2B1 silencing led to a significant decrease of cancer stem cell viability compared with the control, while the cell viability of the rescue of hnRNP A2B1’s expression was similar to that of the control (hnRNP A2B1-scambled) (Fig. 2 C). At the same time, the apoptotic activity of cancer stem cells transfected with hnRNP A2B1-siRNA or hnRNP A2B1-siRNA-2 was significantly increased (Fig. 2 D), indicating that the hnRNP A2B1 silencing promoted apoptosis of cancer stem cells. The rescue of hnRNP A2B1’s expression generated the similar result to the control (Fig. 2 D). The analysis of cell cycle showed that the hnRNP A2B1 knockdown induced the cell cycle arrest in G2 phase of melanoma stem cells, while the rescue of hnRNP A2B1’s expression yielded the similar result to the control (Fig. 2 E). These data revealed that the hnRNP A2B1 silencing triggered the cell cycle arrest, leading to apoptosis of melanoma stem cells. Underlying mechanism of hnRNP A2B1 on the stemness of melanoma stem cells To reveal the hnRNP A2B1-mediated regulatory mechanism of the stemness of melanoma stem cells, RNA immunoprecipitation (RIP) assay and RNA sequencing were conducted in melanoma stem cells (MDA-MB-435) to identify the RNAs interacted with hnRNP A2B1 on a genome-wide scale. The RNA sequencing data were deposited in National Center for Biotechnology Information (NCBI) with an accession no. PRJNA658448. RNA-seq results demonstrated that the expression levels of 36 and 69 mRNAs bound to the hnRNP A2B1 protein were significantly increased and decreased in melanoma stem cells compared with melanoma non-stem cells, respectively (Fig. 3 A and Table 1 ). Due to their importance, the upregulated mRNAs bound to the hnRNP A2B1 protein were further characterized. To confirm the results, the expression levels of six mRNAs, selected from the 36 upregulated mRNAs, were examined. The quantitative real-time PCR data showed that the six mRNAs bound to hnRNP A2B1 were significantly upregulated in melanoma stem cells (MDA-MB-435 and A375) (Fig. 3 B), confirming the RIP data. Table 1 The differential mRNAs bound to the hnRNP A2B1 protein in melanoma stem cells and non-stem cells. mRNAs upregulated in melanoma stem cells mRNAs downregulated in melanoma stem cells MT-CO1, MT-CO3, SGCD, SCLT1, MT-ND4, CNTNAP5, DOCK2, PPARA, TPPP3, ANKRD13C, EIF3H, RNF128, FHOD3, AC099518.3, DCT, FAM196B, FAM102B, DTNA, CDH18, CTAGE5, BANP, TRIM44, MT-ND4L, APAF1, BAHCC1, AF130351.1, NELL2, DAP, EEF2, SMIM28, ZDHHC1, JAZF1, F11, ZNF200. ANXA4, ADD3, ADAMTSL1, B3GAT2, ERI3, TNRC6C, XRCC1, PKMYT1, CYB5R4, NIN, MPRIP, ST8SIA5, PLEKHD1, YEATS2, HBE1, ADRA1B, FOXP1, ZCCHC12, NMNAT3, JTB, TAF15, INTS13, TP53BP1, B4GALNT3, UGP2, TRPM2, KBTBD8, ANKRD33B, RNF165, EML5, FGF14, FBLN1, MSRB3, TCF12, HBG2, FOXN2, KRTAP8-1, LOXHD1, NUP88, RTN1, L34079.1, TDP1, UBE2NL, PPP1R9B, TAL1, STXBP4, LAMA5, VWC2L, TMEM106C, CDH8, KCNK10, GRB10, GRIK2, TMEM132C, CDH2, ERC2, GYG2, XG, SLC6A4, SKOR1, TTR, HGD, C4orf50, AC010547.4, MAP2K5, CDC14B, MYO9A, ERG, MRPL46. KEGG analysis indicated that the upregulated genes interacting with the hnRNP A2B1 protein were involved in cellular pathways, including apoptosis (Fig. 3 C). The above results showed that the hnRNP silencing led to apoptosis of melanoma stem cells. Thus apoptosis was further investigated. Among the 36 upregulated RNAs in melanoma stem cells, TPPP3 (tubulin polymerization promoting protein family member 3) [ 27 ], DOCK2 (dedicator of cytokinesis 2) [ 28 ], EIF3H (eukaryotic translation initiation factor 3 subunit H) [ 29 ], RNF128 (ring finger protein 128) [ 30 ], DAPK1 (death-associated protein kinase 1) [ 31 ] and SYT7 (synaptotagmin 7) [ 32 ] were reported to be associated with apoptosis. Therefore, the effects of hnRNP A2B1 on the splicing of these six genes were explored. The RIP data analysis revealed that the intron sequences of all 6 genes were found in the RIP products (Fig. 3 D), showing that the precursor mRNAs of 6 genes were bound to hnRNP A2B1 and then spliced in the hnRNP A2B1 complex. The analysis of the eCLIP (cross-linking immunoprecipitation) data in the encyclopedia of DNA elements (ENCODE) project ( https://www . encodeproject.org) using USCS tool (Gene Interaction) revealed that hnRNP A2B1 was interacted with EIF3H, which was consistent with our results. The results showed that the sequences of 6 genes bound to hnRNP A2B1 were different in melanoma stem cells and non-stem cells (Fig. 3 D), suggesting the difference of hnRNP A2B1-mediated splicing between melanoma stem cells and non-stem cells. To confirm the hnRNP-mediated splicing of TPPP3, EIF3H, DOCK2, DAPK1, RNF128 and SYT7 in melanoma stem cells, melanoma stem cells were transfected with hnRNP A2B1-siRNA or hnRNP A2B1-siRNA-scrambled,followed by the extraction of total RNAs and then the detection of a randomly selected intron of a gene with semi-quantitative RT-PCR. The results demonstrated that the intron content was obviously decreased in the hnRNP A2B1-siRNA-transfected melanoma stem cells compared with the hnRNP A2B1-siRNA-scrambled treatment (Fig. 3 E). These data indicated that hnRNP mediated the splicing of TPPP3, EIF3H, DOCK2, DAPK1, RNF128 and SYT7 in melanoma stem cells. To explore the influence of hnRNP A2B1 on the expressions of TPPP3, EIF3H, DOCK2, DAPK1, RNF128 and SYT7 in melanoma stem cells, hnRNP A2B1 was silenced or overexpressed, followed by the examination of gene expression. Western blot data indicated that the hnRNP A2B1 silencing led to significant downregulations of TPPP3, EIF3H and DOCK2 and upregulations of DAPK1, RNF128 and SYT7 in melanoma stem cells compared with the control (Fig. 3 F). On the other hand, the hnRNP A2B1 overexpression resulted in significant upregulations of TPPP3, EIF3H and DOCK2 and downregulations of DAPK1, RNF128 and SYT7 in melanoma stem cells (Fig. 3 G). These data indicated that promoted the expressions of EIF3H, TPPP3 and DOCK2 and inhibited the expressions of DAPK1, SYT7 and RNF128 in melanoma stem cells. Collectively, it could be concluded that hnRNP A2B1 promoted tumorigenesis of melanoma stem cells via regulating the splicing of the precursor mRNAs of TPPP3 , DOCK2 , EIF3H , RNF128 , DAPK1 and SYT7 . Roles of TPPP3, DOCK2, EIF3H, RNF128, DAPK1 and SYT7 in apoptosis of melanoma stem cells To reveal the roles of TPPP3, DOCK2, EIF3H, RNF128, DAPK1 and SYT7, which were bound to with hnRNP A2B1, in apoptosis of melanoma stem cells, the expression levels of these genes were examined. Northern blot data indicated that TPPP3, DOCK2 and EIF3H were significantly upregulated in melanoma stem cells compared with cancer non-stem cells, while RNF128, DAPK1 and SYT7 were downreglated in melanoma stem cells (Fig. 4 A). Western blots essentially yielded the similar results (Fig. 4 B). These data suggested that TPPP3, DOCK2, EIF3H, RNF128, DAPK1 and SYT7 played important roles in apoptosis of melanoma stem cells. To explore the effects of TPPP3, DOCK2, EIF3H, RNF128, DAPK1 and SYT7 on apoptosis of melanoma stem cells, the expressions of these genes were respectively silenced by sequence-specific siRNAs, followed by caspase 3/7 detection. The results of quantitative real-time PCR and Western blots showed that the expression of TPPP3, DOCK2, EIF3H, RNF128, DAPK1 or SYT7 was silenced by sequence-specific siRNA compared with the control (Fig. 4 C and 4 D). The knockdown of EIF3H, TPPP3 or DOCK2 promoted apoptosis of stem cells (Fig. 4 E). However, the RNF128, DAPK1 or SYT7 silencing had no effect on apoptosis of melanoma stem cells (Fig. 4 E). On the other hand, the overexpression of RNF128, DAPK1 or SYT7 led to a significant increase of apoptosis of melanoma stem cells, while the TPPP3, DOCK2 or EIF3H overexpression had no effect on apoptosis of melanoma stem cells (Fig. 4 F and G). Taken together, hnRNP A2B1 regulated the pre-mRNA splicing of pro-apoptosis genes (DAPK1, SYT7 and RNF128) and anti-apoptosis genes (EIF3H, TPPP3 and DOCK2), thus leading to the suppression of apoptosis of cancer stem cells (Fig. 4 H). Role of hnRNP A2B1 in tumorigenesis of melanoma stem cells in vivo To evaluate the impact of hnRNP A2B1 on tumorigenesis of melanoma stem cells in vivo , melanoma stem cells (A375 and MDA-MB-435) transfected with hnRNP A2B1-shRNA or hnRNP A2B1-shRNA-scrambled were injected into nude mice, followed by tumor examination (Fig. 5 A). The expression of hnRNP A2B1 could be stably silenced by hnRNP A2B1-shRNA. The results showed that the tumor sizes were significantly reduced in mice treated with hnRNP A2B1-shRNA compared with those in mice treated with hnRNP A2B1-shRNA-scrambled (Fig. 5 B), indicating that the hnRNP A2B1 shRNA suppressed tumor development in vivo . The data of tumor volume revealed that the knockdown of hnRNP A2B1 significantly decreased tumor volume compared with the control (Fig. 5 C). At the same time, the hnRNP A2B1 silencing led to significant decreases of tumor weights compared with the control (Fig. 5 D). Western blot data revealed that the hnRNP A2B1 knockdown significantly decreased the expressions of stemness genes sox2, oct4 and ALDH1 in solid tumors (Fig. 5 E). These results demonstrated that hnRNP A2B1 could promote tumorigenesis of melanoma stem cells in vivo. To evaluate whether the suppression of tumorigenesis resulted from the downregulation of hnRNP A2B1 and its target genes, the expression levels of hnRNP A2B1 and its targets in solid tumors of mice treated with hnRNP A2B1-shRNA or hnRNP A2B1-shRNA-scrambled were examined. The quantitative real-time PCR analysis showed that the hnRNP A2B1 RNA expression was significantly reduced in the solid tumors of the hnRNP A2B1-shRNA-treated mice compared with the control (Fig. 5 F). Western blots yielded essentially the similar results (Fig. 5 G). To explore the effects of hnRNP A2B1 silencing on its targets (TPPP3, DOCK2, EIF3H, RNF128, DAPK1 and SYT7) in vivo , the protein levels of 6 genes in solid tumors were examined. Western blot results showed that the expressions of TPPP3, EIF3H and DOCK2 were significantly decreased in the solid tumors of mice treated with hnRNP A2B1shRNA compared with the controls, while the SYT7, DAPK1 and RNF128 protein levels were significantly increased (Fig. 5 H). These data indicated that hnRNP A2B1 could affect the splicing of TPPP3 , DOCK2 , EIF3H , RNF128 , DAPK1 and SYT7 in vivo . To evaluate the influence of hnRNP A2B1 knockdown on apoptosis in vivo , immunohistochemistry analysis of solid tumors was conducted. The results revealed that the hnRNP A2B1 silencing resulted in the decreased expression of the proliferation marker Ki-67 and the increased expression of caspase 3 compared with the control (Fig. 5 I), indicating that the hnRNP A2B1 silencing triggered apoptosis of melanoma stem cells in vivo . Taken together, the findings revealed that hnRNP A2B1 played a positive role in tumorigenesis of melanoma stem cells in vivo by affecting the splicing of TPPP3 , DOCK2 , EIF3H , RNF128 , DAPK1 and SYT7 , thus suppressing apoptosis of melanoma stem cells. Discussion Apoptosis or programmed cell death, an evolutionarily conserved process in organisms, plays very important roles in tumorigenesis [ 33 ]. At present, apoptosis evading is known as a hallmark of cancers [ 34 ]. Tumor cells have developed various strategies to evade apoptosis in the key modulators of apoptosis pathways [ 35 ]. As well known, the process of apoptosis is controlled by many genes that are differentially expressed in cancers. Alternative splicing is one of the crucial causes for the differential expression of multiple genes [ 11 ]. It is reported that the formation of hnRNP assemblies on pre-mRNA promotes the following alternative splicing events [ 36 , 37 ]. Many hnRNPs have been shown to bind to some specific RNAs and then regulate the post-transcriptional processing of RNAs, thus affecting the expression of RNAs. HnRNP L can directly target the immediate downstream 5’ splice-sites of its binding sequence, and subsequently regulate a further downstream exon 5’ splice-sites selection [ 38 ]. HnRNP A1 can bind to the stem loop of pri-miRNA-18a and modify the secondary structure of this RNA, thereby generating a more favorable cleavage site for Drosha [ 39 ]. During the process of influenza A virus (IAV) infection, host hnRNP K binds to virus mRNA and promotes U1 snRNP recruitment, resulting in mRNA mis-splicing to prevent IAV replication [ 40 ]. In the past decades, some hnRNPs are found to be involved in the RNA splicing. However, the relationship between hnRNP-mediated RNA splicing and apoptosis of cancer stem cells has not been explored. In this study, based on the analysis of 18 human hnRNPs, it was found that hnRNP A2B1, upregulated in melanoma stem cells, could suppress apoptosis of melanoma stem cells via post-transcriptional regulation. HnRNP A2B1 takes effects on mRNA stability, mRNA transport, mRNA alternative splicing, cellular senescence and telomere stability by binding single-stranded DNA [ 41 – 43 ]. However, the function of hnRNP A2B1 in cancer stem cells remains unclear. As reported, hnRNP A2B1 is required for the alternative splicing of apoptosis-related genes, such as the tumor suppressor BIN1 and the anti-apoptotic gene CFLAR (c-FLIP) [ 44 ]. In glioblastoma, breast cancer and pancreatic cancer, hnRNP A2B1 acts as an oncogene by regulating the splicing of apoptosis-associated genes to inhibit apoptosis of cancer cells [ 44 – 46 ]. These results are consistent with our findings. In this context, our findings provided novel insights into the role of hnRNP-mediated RNA splicing in apoptosis of melanoma stem cells. As a splicing factor, hnRNPs can bind to RNAs to mediate the mRNAs’ transcriptional processing, thereby regulating the expression levels of multiple genes [ 47 ]. It can regulate mRNA localization [ 48 ], mRNA stability [ 49 ] and mRNA deadenylation [ 41 ]. At present, however, the mechanism of hnRNP-mediated apoptosis of cancer stem cells remains unknown. In this study, the results showed that hnRNP A2B1 suppressed apoptosis of melanoma stem cells through post-transcriptional regulation of apoptosis-related DAPK1 , SYT7 , RNF128 , EIF3H , TPPP3 and DOCK2 genes. As reported, TPPP3, DOCK2 and EIF3H act as oncogenes through suppressing apoptosis of cancer cells [ 50 – 52 ], while RNF128, DAPK1 and SYT7 function as tumor suppressors via promoting apoptosis of many types of cancer cells [ 53 , 54 ]. Our findings revealed that the hnRNP A2B1-mediated splicing triggered the upregulation of TPPP3, DOCK2 and EIF3H, and the downregulation of RNF128, DAPK1 and SYT7 in melanoma stem cells, leading to the suppression of apoptosis of cancer stem cells. It has been found that the hnRNP A2B1-mediated splicing can increase or decrease the expression levels of its target genes by interacting with other factors [ 55 , 56 ]. In this context, our study revealed a novel mechanism of hnRNP A2B1-mediated suppression of apoptosis of melanoma stem cells. HnRNP A2B1 protein might be a target for melanoma therapy. Except for the apoptosis-associated genes, the other genes of our RNA immunoprecipitation assay, which were upregulated in melanoma stem cells, merited to be further investigated. The proteins interacted with hnRNPA2B1 could be characterized in the future work. Conclusion Our findings revealed that the hnRNP A2B1-mediated splicing triggered the upregulation of TPPP3, DOCK2 and EIF3H, and the downregulation of RNF128, DAPK1 and SYT7 in melanoma stem cells, leading to the suppression of apoptosis of cancer stem cells. HnRNP A2B1 protein might be a target for melanoma therapy. Abbreviations AEC: 3-amino-9-ethylcarbazole bFGF: basic fibroblast growth factor BWA: Burrows Wheeler Aligner CSCs: cancer stem cells DAB: diaminobenzidine DAPK1: death-associated protein kinase 1 DOCK2: dedicator of cytokinesis 2 EDTA: ethylene diamine tetraacetic acid EGF: epidermal growth factor EIF3H: eukaryotic translation initiation factor 3 subunit H GFP: green fluorescence protein GO: gene ontology HEPES: N-2-hydroxyethylpiperazine-N-ethane-sulphonicacid hg38: human reference 38 hnRNPs: heterogenous ribonuclear proteins IACUC: Institutional Animal Care and Use Committee IAV: influenza A virus IGV: Integrative Genomics Viewer KEGG: Kyoto encyclopedia of genes and genomes miRNAs: microRNAs NCBI: National Center for Biotechnology Information PBS: phosphate buffer saline pre-mRNAs: precursor mRNAs PVDF: polyvinylidene difluoride RBPs: RNA-binding proteins RIP: RNA immunoprecipitation RNAi: RNA interference RNF128: ring finger protein 128 RNP: ribonucleoprotein SDS: sodium dodecyl sulfate shRNA: short hairpin RNA snRNP: small nuclear RNP SYT7: synaptotagmin 7 TBS: triethanolamine-buffered saline solution TPPP3: tubulin polymerization promoting protein family member 3 Declarations Ethics approval and consent to participate All procedures conducted on mice in this study were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC). The animal study was reviewed and approved by the Institutional Animal Care and Use Committees of Zhejiang University (Approval No.14843) Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests Funding This work was financially supported by the National Key Research and Development Program of China (2018YFC0310703) and China Ocean Mineral Resources R & D Association (DY135-B-04). Authors' contributions XZ conceived of the study conception and designed the work. MC and HW performed all experiments, interpreted data and drafted the work. MC and XZ wrote the manuscript. All authors have read and approved the final manuscript. Acknowledgements Not applicable. References Plaks V, Kong N, Werb Z. The cancer stem cell niche: how essential is the niche in regulating stemness of tumor cells. Cell stem cell. 2015;16(3):225–38. Vlashi E, Pajonk F. Cancer stem cells, cancer cell plasticity and radiation therapy. 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Tumor Biology. 2017;39(3):1010428317694318. Kutluay SB, Emery A, Penumutchu SR, et al. Genome-wide analysis of heterogeneous nuclear ribonucleoprotein (hnRNP) binding to HIV-1 RNA reveals a key role for hnRNP H1 in alternative viral mRNA splicing. J Virol. 2019;93(21):e01048-19. Gordon H, Ajamian L, Valiente-Echeverrìa F, et al. Depletion of hnRNP A2/B1 overrides the nuclear retention of the HIV-1 genomic RNA. RNA Biol. 2013;10(11):1714–25. Yang Y, Wei Q, Tang Y, et al. Loss of hnRNPA2B1 inhibits malignant capability and promotes apoptosis via down-regulating Lin28B expression in ovarian cancer. Cancer Lett. 2020;475:43–52. Wang X, Wang H, Zhao S, et al. Eukaryotic translation initiation factor EIF3H potentiates gastric carcinoma cell proliferation. Tissue Cell. 2018;53:23–9. Guo X, Chen SY. Dedicator of cytokinesis 2 in cell signaling regulation and disease development. J Cell Physiol. 2017;232(8):1931–40. Ye K, Li Y, Zhao W, et al. Knockdown of tubulin polymerization promoting protein family member 3 inhibits cell proliferation and invasion in human colorectal cancer. J Cancer. 2017;8(10):1750–8. Chen YC, Chan JY, Chiu YL, et al. Grail as a molecular determinant for the functions of the tumor suppressor p53 in tumorigenesis. Cell Death Differ. 2013;20(5):732–43. Steinmann S, Kunze P, Hampel C. DAPK1 loss triggers tumor invasion in colorectal tumor cells. Cell Death Dis. 2019;10(12):1–19. Chen ZY, Cai L, Zhu J, et al. Fyn requires HnRNPA2B1 and Sam68 to synergistically regulate apoptosis in pancreatic cancer. Carcinogenesis. 2011;32(10):1419–26. Liang S, Yu B, Qian D, et al. Human cytomegalovirus ie2 affects the migration of glioblastoma by mediating the different splicing patterns of RON through hnRNP A2B1. Neuroreport. 2019;30(12):805–11. Supplementary Tables Table S1. The primer sequences used for quantitative real-time PCR. Gene Primer hnRNP A2B1 5’-GGCAGCATCAACCTCAGCCAT-3’ 5’-ACTGTTCCTTTGGAGAGG-3’ hnRNP C 5’-TGTTTCATAGCATGCGGCAC-3’ 5’-TGGGCTCCA AGGGTAGTTCA-3’ hnRNP D 5’-GCGTGGGTTCTGCTTTATTACC-3’ 5’-TT GCTGATATTGTTCCTTCGACA-3’ hnRNP E1 5’-CCACGTAACGAGCCCAACT -3’ 5’-TTCCGTGCATAAGAAGCCGA-3’ hnRNP E2 5’-ACTGGAAGAGGAC ATAAGCA-3’ 5’-CCAATGAGAGA-GCCACAC-3’ hnRNP F 5’-CTGCTCTGT TGAGGACGTG-3’ 5’-CCTGCCCTCTCTAGTGTAGATG-3’ hnRNP G 5’-A CATGAATGGAAAGCTCCTGT-3’ 5’-CGTGGTGGTGGTGCATAATC-3’ hnRNP H1 5’-AAGGAAAGAATAGGGCACA-3’ 5’-CTCCACCATAAGCACC AC-3’ hnRNP H2 5’-AGTGATGCGCTTCTTCTCTGA-3’ 5’-TTCGGACCTG TATGCTTCAAC-3’ hnRNP I 5’-CCACGTAACGAGCCCAACT-3’ 5’-TTCCG TGCATAAGAAGCCGA-3’ hnRNP K 5’-CGCTCGTTTTCTGTCTAGCTC-3’ 5’-ACGGGCACACCAATCAGTTA-3’ hnRNP L 5’-TACGCAGCCGACAACCAA ATA-3’ 5’-CTCCGGGAGTCATCCGAGT-3’ TPPP3 5’-GTCTGTCACCTCTT CATGCA-3’ 5’-CAGGAGAGCAGAAATCACAC-3’ DOCK2 5’-CTTGGAGG TCCTCAGCTGTC-3’ 5’-GTCTGAGCTGGTCTGGAAGG-3’ EIF3H 5’-GTG CTTTTGGGTCTGGTTGT-3’ 5’-ATA-CCAGCCCACGTGAAGAT-3’ RNF128 5’- GGGAATT-GAGGTGGATGTTG-3’ 5’-GTGGCTCATCTGCTCCTTGT-3’ DAPK1 5’-GATAGAAATGTCCCCAAACCTCG-3’ 5’-TCTTCTTTGGATCCT TGACCAGAA-3’ SYT7 5’-ACTC-CATCATCGTGAACATCATC-3’ 5’-TCGA AGGCGAAGGACTCATTG-3’ MT-CO1 5’-GAGCTGCTGTT-CGGTGTCC-3’ 5’-TGCCAGTGGTAGAGATGGTTG-3’ GAPDH 5’-GGTATCGTGGAAG GACT CATGAC-3’ 5’ATGCCAGTGAGCTTCCCGTTCAG-3’ Table S2. The probes used for Northern blot. Probe Sequence hnRNP A2B1 5’-CTTCCACTCCTAGAACTCTGAACTTCCTGC-3’ TPPP3 5’-GCATGGCAGCGAGCACAGACATGG-3’ EIF3H 5’-GGAAATGATGCGGAGCCTTCGCCAT-3’ DAPK1 5’-CAACATCATGCAAAGTGAAACAGT-3’ SYT7 5’-ATTGATAGCCTTCTTCTCACTGCGC-3’ DOCK2 5’-GCTTCTCTCAGGAACCTTACCCAAG-3’ RNF128 5’-GCAATAGAGACATGTGAATGTGGCG-3’ Table S3. The primers used for semi-quantitative RT-PCR. Intron Primer sequence TPPP3 5’-AACCCTCTTCCACCAAAAAC-3’ 5’-AAGCACTGGGCGGAT-3’ DOCK2 5’-GTGGACAAATGGGTGAAC-3’ 5’-CGCATCTCTCATCTCATCC-3’ EIF3H 5’-AACTCCCTTTACCTTACTAG-3’ 5’-ATTTCTCACTGTCTTCTGCT-3’ RNF128 5’-CAGAGGTTATGTTGCTAG-3’ 5’-TAAATCACAACAGCATCAGC-3’ DAPK1 5’-AACTCCCTTTACCTTACTAG-3’ 5’-ATTTCTCACTGTCTTCTGCT-3’ SYT7 5’-CAAACTACCCACCACGCACT-3’ 5’-CTAAGACAAAGTGAGGCAGC-3’ β-tubulin 5’-GCCAGATGCCAAGTGACAAG-3’ 5’-TAGTATTCCTCTCCTTCTTC-3’ Table S4. The siRNA sequences used for gene silencing. siRNA sequence hnRNP A2B1-siRNA 5’-AGGAACAGUUCCGUAAGCUCUUUAU-3’ hnRNP A2B1-siRNA-2 5’-CGUGCUGTUGCUUGUGUGG-3’ TPPP3-siRNA 5’-CCGGGCCAAUGUGGGCGUCACUAAA-3’ DOCK2-siRNA 5’-AGGAAGUGACAGUUGAGAA-3’ EIF3H-siRNA 5’-GCAACTCTTGGAAGAAATATACTCG-3’ RNF128-siRNA 5’-GAGGCAAUUAAAGGCAGAU-3’ DAPK1-siRNA 5’-CCGGCCACGUCGAUACCUUG-3’ SYT7-siRNA 5’-UCACCGUGAAGAUCAUGAA-3’ siRNA-scrambled 5’-CCGGGCGCGAUAGCGCUAAUA-3’ Table S5. The primer sequences used for gene overexpression. Gene Primer sequence hnRNP A2B1 primer 1 5’-GCGGAATTCATGGAGAAAACTTTAGAAAC-3’ hnRNP A2B1 primer 2 5’-ATACTCGAGTCAGTATCGGCTCCTCCCAC-3’ hnRNP A2B1 primer 3 5’-GGAACAGTTCCGTAAGCACTTTATTGGTGG-3’ hnRNP A2B1 primer 4 5’-CCACCAATAAAGTGCTTACGGAACTGTTCC-3’ TPPP3 5’-ATAGAATTCATGGCAGCGAGCACAGACAT-3’ 5’-GCGCTCGAGTCACTTCTTCACCTTGGCAT-3’ DOCK2 5’-ATAGATATCATGGCCCCCTGGCGCAAAGC-3’ 5’-TATCTCGAGTCACAGGTCCGTGGACAGCG-3’ EIF3H 5’-ATAGAATTCATGGCGTCCCGCAAGGAAGG-3’ 5’-GCGCTCGAGTTAGTTGTTGTATTCTTGAA-3’ RNF128 5’-ATAGAATTCATGGGGCCGCCGCCTGGGGC-3’ 5’-GCGCTCGAGTTAAGATTTAATTTCTCGAA-3’ DAPK1 5’-TATGAATTCATGACCGTGTTCAGGCAGGA-3’ 5’-ATAGCGGCCGCTCACCGGGATACAACAGAGC-3’ SYT7 5’-TATAAGCTTATGT ACCGGGACCCGGAGGC-3’ 5’-TATCTCGAGTCAGGCCTTCAGCTGGTGCC-3’ Cite Share Download PDF Status: Published Journal Publication published 28 Jan, 2021 Read the published version in Stem Cell Research & Therapy → Version 1 posted Review # 3 received at journal 05 Nov, 2020 Editorial decision: Major Revision 05 Nov, 2020 Reviewer # 4 agreed at journal 27 Oct, 2020 Reviewer # 3 agreed at journal 25 Oct, 2020 Review # 2 received at journal 22 Oct, 2020 Review # 1 received at journal 20 Oct, 2020 Reviewer # 1 agreed at journal 17 Oct, 2020 Reviewer # 2 agreed at journal 17 Oct, 2020 Editor assigned by journal 16 Oct, 2020 Reviewers invited by journal 16 Oct, 2020 Editor invited by journal 15 Oct, 2020 Submission checks completed at journal 14 Oct, 2020 First submitted to journal 13 Oct, 2020 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-92870","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":3595187,"identity":"1e01ec75-f751-48a6-b11d-787f106c6c76","order_by":0,"name":"Mengqi Chu","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mengqi","middleName":"","lastName":"Chu","suffix":""},{"id":3595188,"identity":"62fcda16-aa86-4dd7-9e76-1ef65b525762","order_by":1,"name":"Haitao Wan","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Haitao","middleName":"","lastName":"Wan","suffix":""},{"id":3595189,"identity":"b06c886d-e0ef-4506-ae23-04e45dfd5597","order_by":2,"name":"Xiaobo Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIie3RsQrCMBCA4SuBdjnpGhHaVzgpdNGHaZd2Kjg5VwSnPkDAwWfwDSKBTqWzg4N9g4wOilYnpxo3wfzbwX0cJAA220+GIDXNAwDZD64ZcTqxyKKvCItQq7R8DSYkFEU9FsTy3aoh0EsF/rYcJk7VZlyTW6zKhhzRKuAnOUyYV8X9FSzW0BAbbRQQT4aJyzCeIPHcfZKbCUGviiIkSvBJHBPCsU07QclUQL04VG2O/PiB9C8mpb7ew1Co/fmynAW++EDe7snXZ6Lpfp9ffrFss9lsf9UDl6dBsCAjDC0AAAAASUVORK5CYII=","orcid":"","institution":"Zhejiang University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xiaobo","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2020-10-14 22:28:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-92870/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-92870/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13287-020-02124-5","type":"published","date":"2021-01-28T15:02:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":3094891,"identity":"7319dcfa-8e32-4db5-ab46-10568e5200b0","added_by":"auto","created_at":"2020-10-20 22:00:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":470425,"visible":true,"origin":"","legend":"Differentially expressed hnRNPs in melanoma stem cells and non-stem cells. (A) Differential expressions of hnRNPs in melanoma stem cells and non-stem cells. Quantitative real-time PCR was used to examine the expression profiles of hnRNP genes (*, p\u003c0.05; **, p\u003c0.01). (B) Western blot analysis of hnRNPs in melanoma (MDA-MB-435) stem cells and non-stem cells. β-tubulin was used as a control. (C) Western blot of hnRNP A2B1 in melanoma (A375) stem cells and non-stem cells. All the experiments were biologically repeated for three times.","description":"","filename":"Online1.Png","url":"https://assets-eu.researchsquare.com/files/rs-92870/v1/9f052f4140df543f5abfd865.Png"},{"id":3094892,"identity":"ce40a2fd-3f38-4b7e-94da-98ed64148e9e","added_by":"auto","created_at":"2020-10-20 22:00:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":656522,"visible":true,"origin":"","legend":"Requirement of hnRNP A2B1 for melanoma stem cells. (A) Knockdown and rescue of hnRNP A2B1 expression in cancer stem cells. Melanoma stem cells were transfected with hnRNP A2B1-siRNA or hnRNP A2B1-siRNA-2. To rescue the expression of hnRNP A2B1 in the hnRNP A2B1-silenced melanoma stem cells, the cells were co-transfected with hnRNP A2B1-siRNA and the plasmid expressing hnRNP A2B1. As a control, hnRNP A2B1-siRNA-scrambled was included in the transfection. At different times after transfection, the hnRNP A2B1 mRNA was detected with Northern blot analysis. β-tubulin was used as a control. (B) Western blot analysis of hnRNP A2B1 silencing and rescue in melanoma stem cells. (C) Impact of hnRNP A2B1 silencing and rescue on the proliferation of melanoma stem cells. Cell viability was evaluated at 36 h after transfection of melanoma stem cells with hnRNP A2B1-siRNA, hnRNP A2B1-siRNA-2, hnRNP A2B1-siRNA and the plasmid expressing hnRNP A2B1 or hnRNP A2B1-siRNA-scrambled (**, p\u003c0.01). (D) Influence of hnRNP A2B1 knockdown and rescue on apoptosis of melanoma stem cells. Melanoma stem cells were treated with hnRNP A2B1-siRNA, hnRNP A2B1-siRNA-2, hnRNP A2B1-siRNA and the plasmid expressing hnRNP A2B1 or hnRNP A2B1-siRNA-scrambled. At 36 h later after transfection, apoptosis was examined with the caspase 3/7 activity assay (**, p\u003c 0.01). (E) Role of hnRNP A2B1 in the regulation of melanoma stem cell cycle. Melanoma stem cells were treated with hnRNP A2B1-siRNA, hnRNP A2B1-siRNA-2, hnRNP A2B1-siRNA and the plasmid expressing hnRNP A2B1. Thirty six hours later, the cell cycle was evaluated (**, p\u003c0.01). All the experiments were biologically repeated for three times.","description":"","filename":"Online2.Png","url":"https://assets-eu.researchsquare.com/files/rs-92870/v1/cc9d38744a3e25e5b537de7c.Png"},{"id":3094893,"identity":"f2245e2b-5345-430e-bbe7-fd767889b7c4","added_by":"auto","created_at":"2020-10-20 22:00:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":408537,"visible":true,"origin":"","legend":". Underlying mechanism of hnRNP A2B1 on the stemness of melanoma stem cells. (A) Heatmap of the differential mRNAs bound to the hnRNP A2B1 protein in melanoma stem cells and non-stem cells. (B) The expression profiles of mRNAs bound to the hnRNP A2B1 protein in melanoma cancer cells and non-stem cells. Quantitative real-time PCR was conducted to detect the mRNA level (*, p\u003c0.05; **, p\u003c0.01). The experiments were biologically repeated for three times. (C) KEGG classification of the differentially expressed genes. (D) The sequence analysis of RIP in melanoma stem cells and non-stem cells using hnRNP A2B1 antibody. Black boxes and lines represented exons and introns, respectively. (E) The splicing mediated by hnRNP A2B1 in melanoma stem cells. Melanoma stem cells were transfected with hnRNP A2B1-siRNA or hnRNP A2B1-siRNA-scrambled. At 36h after transfection, Total RNAs were extracted and then subjected to semi-quantitative RT-PCR using intron-specific primers. β-tubulin was used as a control. (F) Influence of hnRNP A2B1 silencing on the expressions of TPPP3, DOCK2, EIF3H, RNF128, DAPK1 and SYT7 in melanoma stem cells. At 36 h after transfection of hnRNP A2B1-siRNA or hnRNP A2B1-siRNA-scrambled, the expression levels of 6 proteins were examined using Western blot. β-tubulin was used as a control. The experiments were repeated for three times. (G) Impact of hnRNP A2B1 overexpression on the expressions of TPPP3, DOCK2, EIF3H, RNF128, DAPK1 and SYT7 in melanoma stem cells. At 48 h after the transfection of the recombinant plasmid expressing hnRNP A2B1, the expression levels of 6 proteins were detected using Western blot. The experiments were biologically repeated for three times.","description":"","filename":"Online3.Png","url":"https://assets-eu.researchsquare.com/files/rs-92870/v1/1045932b52158c75c0186871.Png"},{"id":3094894,"identity":"349bd05f-f5a8-48f9-b517-a0e3f210f6b1","added_by":"auto","created_at":"2020-10-20 22:00:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":754728,"visible":true,"origin":"","legend":"Roles of TPPP3, DOCK2, EIF3H, RNF128, DAPK1 and SYT7 in apoptosis of melanoma stem cells. (A) Expression levels of mRNAs in melanoma stem cell and non-stem cells. Northern blot was conducted to detect the mRNA level. β-tubulin was used as a control. (B) Detections of proteins in melanoma stem cells and non-stem cells by Western blot. β-tubulin was used as a control. (C) Silencing of TPPP3, DOCK2, EIF3H, RNF128, DAPK1 or SYT7 in melanoma stem cells. Melanoma stem cells were transfected with sequence-specific siRNA. At 36 h after transfection, the expression level of mRNA was detected using quantitative real-time PCR. As a control, siRNA-scrambled was included in the assays (**, p \u003c0.01). (D) Western blot analysis of gene silencing in melanoma stem cells. (E) Influence of gene silencing on apoptosis of melanoma stem cells. Melanoma stem cells treated with siRNA were subjected to caspase 3/7 activity assay at 36 h after siRNA transfection (*, p\u003c 0.05; **, p \u003c0.01). (F) Overexpression of TPPP3, DOCK2, EIF3H, RNF128, DAPK1 or SYT7 in melanoma stem cells. Melanoma stem cells were transfected with the recombinant plasmid expressing TPPP3, DOCK2, EIF3H, RNF128, DAPK1 or SYT7. As a control, vector alone was included in the transfection. At 48 h after transfection, the proteins were examined with Western blot. (G) Impact of gene overexpression on apoptosis of melanoma stem cells. Melanoma stem cells transfected with a vector expressing a gene were subjected to caspase 3/7 activity assay at 48 h after transfection (**, p \u003c0.01). Vector alone was used as a control. All he experiments were biologically repeated for three times. (H) Mode for the hnRNP A2B1-mediated apoptosis suppression of melanoma stem cells.","description":"","filename":"Online4.Png","url":"https://assets-eu.researchsquare.com/files/rs-92870/v1/e9f0acd15bd6309d940d1971.Png"},{"id":3094895,"identity":"4b424cb7-9f68-433c-9553-2cb119516840","added_by":"auto","created_at":"2020-10-20 22:00:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1261049,"visible":true,"origin":"","legend":"Role of hnRNP A2B1 in tumorigenesis of melanoma stem cells in vivo. (A) A flow diagram of the in vivo experiments. (B) Effects of hnRNP A2B1 knockdown on tumor growth in nude mice injected with melanoma stem cells (A375 or MDA-MB-435). The melanoma stem cells were treated with hnRNP A2B1-shRNA or hnRNP A2B1-shRNA-scrambled. Six weeks later, the nude mice were sacrificed and the solid tumors were observed. (C) Influence of hnRNP A2B1 silencing on tumor volume. The tumor volume of mice treated with hnRNP A2B1-shRNA or hnRNP A2B1-shRNA-scrambled was examined every week. The mean of 5 mice was indicated (**, p\u003c0.01). (D) Impact of hnRNP A2B1 knockdown on tumor weight in nude mice. The solid tumors from nude mice were taken out and weighed (**, p\u003c0.01). (E) The expressions of stemness genes in solid tumors. Western blot was used to examine the expressions of stemness genes in the solid tumors of mice treated with hnRNP A2B1-shRNA or hnRNP A2B1-shRNA-scrambled. β-tubulin was used as a control. (F) Expression level of hnRNP A2B1 in solid tumors of mice. Total RNAs extracted from the solid tumors of hnRNP A2B1-shRNA- or hnRNP A2B1-shRNA-scrambled-treated mice were analyzed using quantitative real-time PCR (**, p\u003c0.01). (G) Protein level of hnRNP A2B1 in the solid tumors of mice treated with hnRNP A2B1-shRNA or hnRNP A2B1-shRNA-scrambled. Western blot analysis was conducted to examine the hnRNP A2B1 protein level. β-tubulin was used as a control. (H) Effects of hnRNP A2B1 silencing on its targets’ expressions in solid tumors. Western blot analysis was conducted to examine the protein levels of hnRNP A2B1’s targets in the solid tumors of mice treated with hnRNP A2B1-shRNA or hnRNP A2B1-shRNA-scrambled. β-tubulin was used as a control. (I) Immunohistological analysis of solid tumors. Solid tumors were analyzed by immunohistochemistry using the antibody against ki67 or Caspase 3 (brown). The nuclei were stained by DPAI (blue). Scale bar, 50μm.","description":"","filename":"Online5.Png","url":"https://assets-eu.researchsquare.com/files/rs-92870/v1/e904e7641498e1e711a91293.Png"},{"id":13604386,"identity":"b015e3a8-a2d1-4042-bcd3-443c28f98f01","added_by":"auto","created_at":"2021-09-17 05:59:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2689258,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-92870/v1/01a84d55-ae4c-46bb-98ae-3cd934903598.pdf"}],"financialInterests":"","formattedTitle":"Requirement of splicing factor hnRNP A2B1 of hnRNPs for tumorigenesis of melanoma stem cells","fulltext":[{"header":"Background","content":" \u003cp\u003eCancer is known as a major cause of death due to its high morbidity and mortality [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. A lot of investigations have suggested that only a small proportion of cancer cells have tumorigenic capability, which is named as cancer stem cells (CSCs) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. CSCs are proved to play essential roles in predicting the biological aggressiveness of cancers. They are correlated with recurrence, metastasis and poor survival in solid tumors [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], thus being responsible for tumorigenesis, tumor differentiation, tumor maintenance, tumor spread, and tumor relapse. CSCs have the ability of unlimited growth and self-renewal [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, elimination of CSCs is difficult but essential to cure cancer completely [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. At present, it is believed that CSCs origin from abnormal expressions of a series of genes. Gene expression regulation plays an essential role in the biogenesis of CSCs. The regulation of gene expression depends on many factors, including DNA sequence, transcriptional regulation, post-transcriptional modification, translational control and protein modification [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Post-transcriptional regulation is the most important regulation process, because it can influence RNA stability, localization, translation efficiency and sequence [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. It also contributes to shaping tissue-type specific proteomes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Post-transcriptional regulation includes transcript stability, binding of RNAs to RNA-binding proteins (RBPs), alternative splicing and regulations by microRNAs (miRNAs) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], thus providing the flexibility for cells to adapt to a wide range of physiological conditions. Among post-transcriptional regulations, alternative splicing is one of the crucial mechanisms for gene expression [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is well known that alternative splicing often results in differential intron and exon retention or skipping and this process can produce different mRNA and protein isoforms from one gene. Studies have shown that over 90% of human RNAs are alternatively spliced, leading to a cellular mRNA accumulation that can encode four to five fold more proteins than protein-coding genes in the genome [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Alternative splicing occurs in a large ribonucleoprotein (RNP) machine called spliceosome [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The spliceosome contains five different small nuclear RNP (snRNP) subunits (U1, U2, U4, U5 and U6) along with more than 200 associated protein cofactors [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The splicing process involves 70\u0026ndash;250 splicing factors depending on the species, which can interact in multiple steps with RNA and proteins [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Among splicing factors, serine/arginine (SR) proteins and heterogenous ribonuclear proteins (hnRNPs) are the crucial families of splicing factors [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. At present, 12 classical SR proteins and 17 canonical hnRNPs are identified [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Most SR proteins act as splicing activators by binding precursor mRNAs (pre-mRNAs) at exonic splicing enhancers. SR proteins often compete with splicing repressors, such as hnRNPs, whose binding to exonic or intronic splicing silencers can inhibit the selection of splicing site, thereby modulating alternative splicing [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. By binding to different splicing factors, pre-mRNAs are able to be alternatively spliced in different ways [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. As reported, some hnRNPs, such as hnRNP A1, K and A2B1, are aberrantly higher expressed in lung cancer, breast cancer or hepatocellular carcinoma [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], suggesting the important roles of hnRNPs in tumorigenesis. However, the influence of hnRNPs on cancer stem cells has not been extensively explored.\u003c/p\u003e \u003cp\u003eTo address this issue, the expression levels of hnRNPs were examined in melanoma stem cells. The results showed that hnRNP A2B1 of 19 hnRNPs was significantly upregulated in melanoma stem cell compared with melanoma non-stem cells. The further investigations revealed that hnRNP A2B1 took great effects on tumorigenesis of melanoma stem cells.\u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMelanoma stem cells and non-stem cells were sorted in our laboratory previously from cell line MDA-MB-435 [24] or A375 [25]. Melanoma stem cells and non-stem cells were cultured in DMEM/F-12 medium (Invitrogen, USA) supplemented with 20 ng/mL epidermal growth factor (EGF) (Beyotime Biotechnology, China), 10 ng/mL basic fibroblast growth factor (bFGF) (Beyotime Biotechnology), 5 mg/mL of insulin (Beyotime Biotechnology), and 2% of B-27 (Sigma, USA) at 37℃ in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification of mRNA with real-time PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNAs were extracted from cells using an RNA Isolation Kit (Ambion, USA). The reverse transcription reaction was conducted with PrimeScript RT Reagent Kit (TaKaRa, Japan). Quantitative real-time PCR was performed using 2\u0026times;ChamQ SYBR qPCR Master Mix (Vazyme, USA). The PCR reaction mixture (10 \u0026mu;L) contained Rox reference Dye, cDNA, ChamQ SYBR qPCR Master Mix (Vazyme) and primers (Table S1). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was included for normalization. The 2\u003csup\u003e-(\u003c/sup\u003e\u003csup\u003e△△\u003c/sup\u003e\u003csup\u003eCt)\u003c/sup\u003e method was used to calculate the relative fold change of mRNA expression [26]. PCR was conducted by maintaining the reaction at 95℃ for 30 s, and then alternating for 40 cycles between 95℃ for 5 s and 60℃ for 30 s.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteins (about 50 \u0026mu;g/protein) were separated using 12% SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with triethanolamine-buffered saline solution (TBS) containing 5% skim milk. Subsequently, the membrane was incubated overnight with a primary antibody, followed by incubation with the alkaline phosphatase-conjugated secondary antibody (Roche, Switzerland) for 2 h at room temperature. After rinses, the membrane was detected with BCIP/NBT substrate (Sangon Biotech, China). All antibodies were purchased from Proteintech Group (USA) at catalog number 67445-1-Ig (hnRNP A2B1), 15057-1-AP (TPPP3), 11310-1-AP (EIF3H), 25136-1-AP (DAPK1), A12757 (SYT7), 66969-1-Ig (DOCK2) and 26015-1-AP (RNF128).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNorthern blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNAs were extracted using a cell/tissue genomic DNA extraction kit (Generay Biotech, China) according to the manufacturer\u0026rsquo;s manual. After electrophoresis of 30 \u0026mu;g RNAs on a 2% agarose gel, the RNAs were transferred to a nylon membrane (Amersham Biosciences, Sweden) for 1 h, followed by ultraviolet cross-linking. The membrane was prehybridized in prehybridization solution (Roche, Switzerland) for 30 min. Subsequently, the membrane was hybridized with a DIG-labeled probe (Table S2) at 42\u003csup\u003e0\u003c/sup\u003eC overnight. The membrane was rinsed and then blocked in a blocking solution (Roche) for 1 h at room temperature. After incubation with the antibody (10 \u0026mu;l) against DIG-labeled alkaline phosphatase (Roche) for 2 h at room temperature, the signals of the membrane were detected with the substrate BCIP/NBT solution (Roche).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA immunoprecipitation (RIP) assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were treated with paraformaldehyde for 10 min at room temperature. After washes with ice-cold phosphate buffer saline (PBS), the cells were incubated with PBS containing 0.125 M glycine for 5 min and then with hypotonic buffer [10 mM N-2-hydroxyethylpiperazine-N-ethane-sulphonicacid (HEPES), 1.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 mM KCl, 0.4% Nonidet P-40, pH 7.9] on ice for 15 min, followed by centrifugation at 3,000 \u0026times;g for 7 min. The pellet was resuspended in sonication buffer [10% sodium dodecyl sulfate (SDS), 0.5 M ethylene diamine tetraacetic acid (EDTA), 1 M Tris-HCl, pH 8.0] and then subjected to ultrasonication. The sample was centrifuged at 12,000 \u0026times;g for 20 s and the supernatant was incubated with antibody-coupled Protein G magnetic beads (70\u0026mu;l) (Bio-Rad Laboratories, USA) at 4℃ overnight. The beads were washed with PBS. Subsequently the RNAs were extracted using RNA Isolation Kit (Ambion, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA-seq and data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe extracted RNAs were subjected to RNA-seq using an Illumina Hiseq 2500 system by Novogene Corporation (China). Briefly, the rRNAs were removed by ribo-zerotm kit (Epicentre, France). Subsequently fragmentation buffer was added to break the RNA into short segments of 250-300bp, followed by the synthesis of cDNAs with random hexamers. After purification with AMPure XP beads, the double-stranded cDNAs were added with A tails and the connection of sequencing joints. The cDNA library was enriched by PCR. Sequencing was performed. After assembly of RNA-seq data, the raw data was processed to remove the sequences of adapter, ploy-N and low-quality reads. The clean reads were aligned to the genome reference consortium human reference 38 (hg38) by BWA (Burrows Wheeler Aligner) and IGV (Integrative Genomics Viewer). Based on the counts of reads, the gene expression profile was obtained.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKyoto encyclopedia of genes and genomes (KEGG) analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe coding sequences of transcripts were extracted and used as queries to search the protein sequences collected in the GO (gene ontology) database with the blast E-value of less than 1\u0026times;10\u003csup\u003e-5\u003c/sup\u003e. The best hit GO identities were assigned to the transcripts. The p-values were corrected for false discovery rate. Deduced genes with homologues in other organisms were used to map to conserved biological pathways.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSemi-quantitative reverse transcription (RT)-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNAs were extracted from cells using a cell/tissue genomic DNA extraction kit (Generay Biotech, China) and then quantified by NanoDrop ND-1000 spectrophotometer. The complementary DNA was synthesized using HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, USA) following the manufacturer\u0026rsquo;s instructions. Subsequently PCR was conducted with sequence-specific primers (Table S3). \u003cem\u003e\u0026beta;-tubulin\u003c/em\u003e was used as a loading control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell viability analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell viability was evaluated using MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (Promega, USA). Cells were seeded onto a 96-well plate. Thirty-six hours later, the plate was incubated with 20 \u0026mu;L of MTS reagent for 1 h at 37℃. Subsequently the absorbance was recorded at 490 nm. All experiments were repeated three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of caspase 3/7 activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCaspase-Glo 3/7 assay (Promega) was used to evaluate the activity of caspase 3/7 according to the manufacturer\u0026rsquo;s protocol. Cells at a density of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e/well were plated onto a 96-well plate. Subsequently 50 mL of caspase-Glo 3/7 reagent (Promega) was added to each well. After incubation in the dark at room temperature for 1 h, the luminescence of cells was measured.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell cycle analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell cycle analysis was conducted with flow cytometry. Cells were fixed in ice-cold ethanol overnight. Then the cells were incubated with DNase-free RNase A (20 mg/mL) for 30 min. After centrifugation at 500\u0026times;g for 5 min, the cells were stained with propidium iodide (PI, 50 mg/mL). The fluorescence intensity of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells was measured with a flow cytometer at an excitation wavelength of 488 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSilencing and overexpression of gene in cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo silence the gene expression in cancer stem cells, RNA interference (RNAi) assay was conducted using gene-specific siRNA (Table S4). The melanoma stem cells (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e) were transfected with 50 nM of siRNA using Lipofectamine 2000 (Invitrogen, USA). All the siRNAs were synthesized by Shanghai GenePharma Co., Ltd. At different time after transfection, the cells were harvested for later use.\u003c/p\u003e\n\u003cp\u003eTo overexpress a gene in cancer stem cells, the gene was amplified using PCR with sequence-specific primers (Table S5), followed by cloning into pcDNA3.1 (+) vector. The recombinant plasmid was transfected into melanoma stem cells using Lipofectamine 2000 (Invitrogen). At different time after transfection, the cells were collected for later use.\u003c/p\u003e\n\u003cp\u003eTo overexpress hnRNP A2B1 in the hnRNP A2B1-silenced melanoma stem cells, hnRNP A2B1 was mutated at a nucleotide (position 32 A\u0026rarr;T) to prevent the recognition by hnRNP A2B1-siRNA. Briefly hnRNP A2B1 was amplified by hnRNP A2B1 primers 1 and 4 (Table S5), and hnRNP A2B1 primers 2 and 3 (Table S5), respectively. Then the amplified products were subjected to PCR with hnRNP A2B1 primers 1 and 2 (Table S5), followed by cloning into pcDNA3.1 (+) vector.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTumorigenicity in nude mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMelanoma stem cells were transfected with hnRNP A2B1-shRNA (short hairpin RNA) (5\u0026rsquo;-AGGAACAGTTCCGTAAGCTCTTTAT-3\u0026rsquo;) to stably silence the expression of hnRNP A2B1. ShRNA was designed with invitrogen BLOCK-iT\u0026trade; RNAi Designer (http://rnaidesigner.thermofisher.com). As a control, the sequence of hnRNP A2B1-shRNA was randomly scrambled, generating hnRNP A2B1-shRNA-scrambled (5\u0026rsquo;-CCGGGCGCGATAGCGCTAATAGCGA-3\u0026rsquo;). ShRNA was cloned into lentiviral vector pLent-U6-GFP-Puro (Vigene Bioscience, USA), followed by transfection into 293T cells using Lipofectamine 2000 reagent (Life Technologies, USA). At 48 h after transfection, the viral particles were collected to infect melanoma stem cells. Subsequently, the cells were cultured in medium contained 10 \u0026micro;g/ml puromycin for three days. After puromycin screening, only the cells expressing green fluorescence\u0026nbsp;protein (GFP) were selected as stable strains expressing shRNA.\u003c/p\u003e\n\u003cp\u003eMelanoma stem cells transfected with hnRNP A2B1-shRNA or hnRNP A2B1-shRNA-scrambled were suspended in physiological saline. Matrigel (Becton, Dickinson and Company, USA) was added to the cell suspension at the final concentration of 33%. Subsequently, 100 \u0026mu;L of the cell suspension was subcutaneously injected into each BALB/c mouse to induce tumor growth. The tumor volume was measured every week. Six weeks later, the mice were sacrificed. The tumor sizes and tumor weights were determined. All procedures conducted on mice in this study were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC). All the methods were carried out in accordance with the approved guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTumor tissue was cut into 5 \u0026mu;m pieces and mounted onto a slide. The slide was dewaxed and hydrated in 100%, 95% and 80% ethanol for 5\u0026thinsp;min, respectively. Subsequently the primary antibody was incubated with the slide. After washing with PBS, the slide was incubated with the secondary antibody for 10\u0026thinsp;min at room temperature. Streptavidin peroxidase was added to the slide, followed by incubation for 10\u0026thinsp;min at room temperature. Then 3-amino-9-ethylcarbazole (AEC) buffer and AEC chromogen (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were mixed and added to the slide. The slide was incubated for 10\u0026thinsp;min at room temperature. The proteins and the nucleus were labeled with diaminobenzidine (DAB) (Sigma, USA) or 4\u0026rsquo;,6-diamidino-2- phenylindole (DAPI), respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe numerical data were analyzed by one-way analysis of variance (ANOVA). The differences between different treatments were analyzed by Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test. All data were presented as mean \u0026plusmn; standard deviation.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003eDifferentially expressed hnRNPs in melanoma stem cells and non-stem cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the roles of hnRNPs in cancer stem cells, the expression levels of 19 hnRNP family genes were characterized in two types of melanoma stem cells and non-stem cells. The melanoma stem cells were previously sorted in our laboratory from melanoma cell lines MDA-MB-435 and A375 [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e], respectively. The results of quantitative real-time PCR revealed that compared with the controls, 3 hnRNPs (A2B1, I and L) and 8 hnRNPs (A2B1, C, E1, H1, H2, I, K and L) were significantly upregulated in MDA-MB-435 stem cells and in A375 stem cells compared with melanoma non-stem cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA), respectively. These data indicated that hnRNP A2B1, hnRNP I and hnRNP L were highly expressed in two types of melanoma cancer stem cells. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB, the expression profiles of hnRNP I and hnRNP L proteins were similar in MDA-MB-435 melanoma stem cells and non-stem cells, while the hnRNP A2B1 protein was significantly upregulated in melanoma stem cells compared with non-stem cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Western blot revealed that the content of hnRNP A2B1 was much higher in A375 melanoma stem cells than that in non-stem cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). Therefore, hnRNP A2B1 was further characterized in melanoma stem cells.\u003c/p\u003e\n\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eRequirement of hnRNP A2B1 for melanoma stem cells\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eTo investigate the role of hnRNP A2B1 in melanoma stem cells, the hnRNP A2B1 expression was knocked down or rescued, followed by the evaluation of stem cell viability. Northern blots showed that the hnRNP A2B1 mRNA was knocked down in melanoma stem cells by hnRNP A2B1-siRNA compared with the control (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). To confirm the efficiency of hnRNP A2B1-siRNA, another siRNA specifically targeting hnRNP A2B1 (hnRNP A2B1-siRNA-2) was transfected into melanoma stem cells. The results indicated that the efficiency of hnRNP A2B1-siRNA-2 was similar to that of hnRNP A2B1-siRNA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). To rescue the expression of hnRNP A2B1 in hnRNP A2B1-silenced melanoma stem cells, the stem cells were co-transfected with hnRNP A2B1-siRNA and the plasmid expressing hnRNP A2B1. The data of Northern blot analysis revealed that the expression of hnRNP A2B1 was rescued in the hnRNP A2B1-siRNA-treated melanoma stem cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Western blot yielded essentially the similar results (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). These data indicated that hnRNP A2B1 was silenced in melanoma stem cells or rescued in the hnRNP A2B1-silenced melanoma stem cells.\u003c/p\u003e\n\u003cp\u003eThe results of MTS assays indicated that the hnRNP A2B1 silencing led to a significant decrease of cancer stem cell viability compared with the control, while the cell viability of the rescue of hnRNP A2B1\u0026rsquo;s expression was similar to that of the control (hnRNP A2B1-scambled) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). At the same time, the apoptotic activity of cancer stem cells transfected with hnRNP A2B1-siRNA or hnRNP A2B1-siRNA-2 was significantly increased (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD), indicating that the hnRNP A2B1 silencing promoted apoptosis of cancer stem cells. The rescue of hnRNP A2B1\u0026rsquo;s expression generated the similar result to the control (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). The analysis of cell cycle showed that the hnRNP A2B1 knockdown induced the cell cycle arrest in G2 phase of melanoma stem cells, while the rescue of hnRNP A2B1\u0026rsquo;s expression yielded the similar result to the control (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE). These data revealed that the hnRNP A2B1 silencing triggered the cell cycle arrest, leading to apoptosis of melanoma stem cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUnderlying mechanism of hnRNP A2B1 on the stemness of melanoma stem cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo reveal the hnRNP A2B1-mediated regulatory mechanism of the stemness of melanoma stem cells, RNA immunoprecipitation (RIP) assay and RNA sequencing were conducted in melanoma stem cells (MDA-MB-435) to identify the RNAs interacted with hnRNP A2B1 on a genome-wide scale. The RNA sequencing data were deposited in National Center for Biotechnology Information (NCBI) with an accession no. PRJNA658448. RNA-seq results demonstrated that the expression levels of 36 and 69 mRNAs bound to the hnRNP A2B1 protein were significantly increased and decreased in melanoma stem cells compared with melanoma non-stem cells, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Due to their importance, the upregulated mRNAs bound to the hnRNP A2B1 protein were further characterized. To confirm the results, the expression levels of six mRNAs, selected from the 36 upregulated mRNAs, were examined. The quantitative real-time PCR data showed that the six mRNAs bound to hnRNP A2B1 were significantly upregulated in melanoma stem cells (MDA-MB-435 and A375) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB), confirming the RIP data.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eThe differential mRNAs bound to the hnRNP A2B1 protein in melanoma stem cells and non-stem cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003emRNAs upregulated in melanoma stem cells\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003emRNAs downregulated in melanoma stem cells\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMT-CO1, MT-CO3, SGCD, SCLT1, MT-ND4, CNTNAP5, DOCK2, PPARA, TPPP3, ANKRD13C, EIF3H, RNF128, FHOD3, AC099518.3, DCT, FAM196B, FAM102B, DTNA, CDH18, CTAGE5, BANP, TRIM44, MT-ND4L, APAF1, BAHCC1, AF130351.1, NELL2, DAP, EEF2, SMIM28, ZDHHC1, JAZF1, F11, ZNF200.\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eANXA4, ADD3, ADAMTSL1, B3GAT2, ERI3, TNRC6C, XRCC1, PKMYT1, CYB5R4, NIN, MPRIP, ST8SIA5, PLEKHD1, YEATS2, HBE1, ADRA1B, FOXP1, ZCCHC12, NMNAT3, JTB, TAF15, INTS13, TP53BP1, B4GALNT3, UGP2, TRPM2, KBTBD8, ANKRD33B, RNF165, EML5, FGF14, FBLN1, MSRB3, TCF12, HBG2, FOXN2, KRTAP8-1, LOXHD1, NUP88, RTN1, L34079.1, TDP1, UBE2NL, PPP1R9B, TAL1, STXBP4, LAMA5, VWC2L, TMEM106C, CDH8, KCNK10, GRB10, GRIK2, TMEM132C, CDH2, ERC2, GYG2, XG, SLC6A4, SKOR1, TTR, HGD, C4orf50, AC010547.4, MAP2K5, CDC14B, MYO9A, ERG, MRPL46.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKEGG analysis indicated that the upregulated genes interacting with the hnRNP A2B1 protein were involved in cellular pathways, including apoptosis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). The above results showed that the hnRNP silencing led to apoptosis of melanoma stem cells. Thus apoptosis was further investigated. Among the 36 upregulated RNAs in melanoma stem cells, TPPP3 (tubulin polymerization promoting protein family member 3) [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e], DOCK2 (dedicator of cytokinesis 2) [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e], EIF3H (eukaryotic translation initiation factor 3 subunit H) [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e], RNF128 (ring finger protein 128) [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e], DAPK1 (death-associated protein kinase 1) [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e] and SYT7 (synaptotagmin 7) [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e] were reported to be associated with apoptosis. Therefore, the effects of hnRNP A2B1 on the splicing of these six genes were explored. The RIP data analysis revealed that the intron sequences of all 6 genes were found in the RIP products (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD), showing that the precursor mRNAs of 6 genes were bound to hnRNP A2B1 and then spliced in the hnRNP A2B1 complex. The analysis of the eCLIP (cross-linking immunoprecipitation) data in the encyclopedia of DNA elements (ENCODE) project (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www\u003c/span\u003e\u003c/span\u003e. encodeproject.org) using USCS tool (Gene Interaction) revealed that hnRNP A2B1 was interacted with EIF3H, which was consistent with our results. The results showed that the sequences of 6 genes bound to hnRNP A2B1 were different in melanoma stem cells and non-stem cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD), suggesting the difference of hnRNP A2B1-mediated splicing between melanoma stem cells and non-stem cells. To confirm the hnRNP-mediated splicing of TPPP3, EIF3H, DOCK2, DAPK1, RNF128 and SYT7 in melanoma stem cells, melanoma stem cells were transfected with hnRNP A2B1-siRNA or hnRNP A2B1-siRNA-scrambled,followed by the extraction of total RNAs and then the detection of a randomly selected intron of a gene with semi-quantitative RT-PCR. The results demonstrated that the intron content was obviously decreased in the hnRNP A2B1-siRNA-transfected melanoma stem cells compared with the hnRNP A2B1-siRNA-scrambled treatment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE). These data indicated that hnRNP mediated the splicing of TPPP3, EIF3H, DOCK2, DAPK1, RNF128 and SYT7 in melanoma stem cells.\u003c/p\u003e\n\u003cp\u003eTo explore the influence of hnRNP A2B1 on the expressions of TPPP3, EIF3H, DOCK2, DAPK1, RNF128 and SYT7 in melanoma stem cells, hnRNP A2B1 was silenced or overexpressed, followed by the examination of gene expression. Western blot data indicated that the hnRNP A2B1 silencing led to significant downregulations of TPPP3, EIF3H and DOCK2 and upregulations of DAPK1, RNF128 and SYT7 in melanoma stem cells compared with the control (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF). On the other hand, the hnRNP A2B1 overexpression resulted in significant upregulations of TPPP3, EIF3H and DOCK2 and downregulations of DAPK1, RNF128 and SYT7 in melanoma stem cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG). These data indicated that promoted the expressions of EIF3H, TPPP3 and DOCK2 and inhibited the expressions of DAPK1, SYT7 and RNF128 in melanoma stem cells.\u003c/p\u003e\n\u003cp\u003eCollectively, it could be concluded that hnRNP A2B1 promoted tumorigenesis of melanoma stem cells via regulating the splicing of the precursor mRNAs of \u003cem\u003eTPPP3\u003c/em\u003e, \u003cem\u003eDOCK2\u003c/em\u003e, \u003cem\u003eEIF3H\u003c/em\u003e, \u003cem\u003eRNF128\u003c/em\u003e, \u003cem\u003eDAPK1\u003c/em\u003e and \u003cem\u003eSYT7\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRoles of TPPP3, DOCK2, EIF3H, RNF128, DAPK1 and SYT7 in apoptosis of melanoma stem cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo reveal the roles of TPPP3, DOCK2, EIF3H, RNF128, DAPK1 and SYT7, which were bound to with hnRNP A2B1, in apoptosis of melanoma stem cells, the expression levels of these genes were examined. Northern blot data indicated that TPPP3, DOCK2 and EIF3H were significantly upregulated in melanoma stem cells compared with cancer non-stem cells, while RNF128, DAPK1 and SYT7 were downreglated in melanoma stem cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Western blots essentially yielded the similar results (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). These data suggested that TPPP3, DOCK2, EIF3H, RNF128, DAPK1 and SYT7 played important roles in apoptosis of melanoma stem cells.\u003c/p\u003e\n\u003cp\u003eTo explore the effects of TPPP3, DOCK2, EIF3H, RNF128, DAPK1 and SYT7 on apoptosis of melanoma stem cells, the expressions of these genes were respectively silenced by sequence-specific siRNAs, followed by caspase 3/7 detection. The results of quantitative real-time PCR and Western blots showed that the expression of TPPP3, DOCK2, EIF3H, RNF128, DAPK1 or SYT7 was silenced by sequence-specific siRNA compared with the control (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). The knockdown of EIF3H, TPPP3 or DOCK2 promoted apoptosis of stem cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE). However, the RNF128, DAPK1 or SYT7 silencing had no effect on apoptosis of melanoma stem cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE). On the other hand, the overexpression of RNF128, DAPK1 or SYT7 led to a significant increase of apoptosis of melanoma stem cells, while the TPPP3, DOCK2 or EIF3H overexpression had no effect on apoptosis of melanoma stem cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF and G).\u003c/p\u003e\n\u003cp\u003eTaken together, hnRNP A2B1 regulated the pre-mRNA splicing of pro-apoptosis genes (DAPK1, SYT7 and RNF128) and anti-apoptosis genes (EIF3H, TPPP3 and DOCK2), thus leading to the suppression of apoptosis of cancer stem cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eH).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRole of hnRNP A2B1 in tumorigenesis of melanoma stem cells \u003cem\u003e\u003cspan class=\"BoldItalic\"\u003ein vivo\u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the impact of hnRNP A2B1 on tumorigenesis of melanoma stem cells \u003cem\u003ein vivo\u003c/em\u003e, melanoma stem cells (A375 and MDA-MB-435) transfected with hnRNP A2B1-shRNA or hnRNP A2B1-shRNA-scrambled were injected into nude mice, followed by tumor examination (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). The expression of hnRNP A2B1 could be stably silenced by hnRNP A2B1-shRNA. The results showed that the tumor sizes were significantly reduced in mice treated with hnRNP A2B1-shRNA compared with those in mice treated with hnRNP A2B1-shRNA-scrambled (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB), indicating that the hnRNP A2B1 shRNA suppressed tumor development \u003cem\u003ein vivo\u003c/em\u003e. The data of tumor volume revealed that the knockdown of hnRNP A2B1 significantly decreased tumor volume compared with the control (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). At the same time, the hnRNP A2B1 silencing led to significant decreases of tumor weights compared with the control (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). Western blot data revealed that the hnRNP A2B1 knockdown significantly decreased the expressions of stemness genes sox2, oct4 and ALDH1 in solid tumors (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE). These results demonstrated that hnRNP A2B1 could promote tumorigenesis of melanoma stem cells \u003cem\u003ein vivo.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate whether the suppression of tumorigenesis resulted from the downregulation of hnRNP A2B1 and its target genes, the expression levels of hnRNP A2B1 and its targets in solid tumors of mice treated with hnRNP A2B1-shRNA or hnRNP A2B1-shRNA-scrambled were examined. The quantitative real-time PCR analysis showed that the hnRNP A2B1 RNA expression was significantly reduced in the solid tumors of the hnRNP A2B1-shRNA-treated mice compared with the control (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF). Western blots yielded essentially the similar results (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG).\u003c/p\u003e\n\u003cp\u003eTo explore the effects of hnRNP A2B1 silencing on its targets (TPPP3, DOCK2, EIF3H, RNF128, DAPK1 and SYT7) \u003cem\u003ein vivo\u003c/em\u003e, the protein levels of 6 genes in solid tumors were examined. Western blot results showed that the expressions of TPPP3, EIF3H and DOCK2 were significantly decreased in the solid tumors of mice treated with hnRNP A2B1shRNA compared with the controls, while the SYT7, DAPK1 and RNF128 protein levels were significantly increased (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH). These data indicated that hnRNP A2B1 could affect the splicing of \u003cem\u003eTPPP3\u003c/em\u003e, \u003cem\u003eDOCK2\u003c/em\u003e, \u003cem\u003eEIF3H\u003c/em\u003e, \u003cem\u003eRNF128\u003c/em\u003e, \u003cem\u003eDAPK1\u003c/em\u003e and \u003cem\u003eSYT7 in vivo\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eTo evaluate the influence of hnRNP A2B1 knockdown on apoptosis \u003cem\u003ein vivo\u003c/em\u003e, immunohistochemistry analysis of solid tumors was conducted. The results revealed that the hnRNP A2B1 silencing resulted in the decreased expression of the proliferation marker Ki-67 and the increased expression of caspase 3 compared with the control (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eI), indicating that the hnRNP A2B1 silencing triggered apoptosis of melanoma stem cells \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eTaken together, the findings revealed that hnRNP A2B1 played a positive role in tumorigenesis of melanoma stem cells \u003cem\u003ein vivo\u003c/em\u003e by affecting the splicing of \u003cem\u003eTPPP3\u003c/em\u003e, \u003cem\u003eDOCK2\u003c/em\u003e, \u003cem\u003eEIF3H\u003c/em\u003e, \u003cem\u003eRNF128\u003c/em\u003e, \u003cem\u003eDAPK1\u003c/em\u003e and \u003cem\u003eSYT7\u003c/em\u003e, thus suppressing apoptosis of melanoma stem cells.\u003c/p\u003e"},{"header":"Discussion","content":" \u003cp\u003eApoptosis or programmed cell death, an evolutionarily conserved process in organisms, plays very important roles in tumorigenesis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. At present, apoptosis evading is known as a hallmark of cancers [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Tumor cells have developed various strategies to evade apoptosis in the key modulators of apoptosis pathways [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. As well known, the process of apoptosis is controlled by many genes that are differentially expressed in cancers. Alternative splicing is one of the crucial causes for the differential expression of multiple genes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It is reported that the formation of hnRNP assemblies on pre-mRNA promotes the following alternative splicing events [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Many hnRNPs have been shown to bind to some specific RNAs and then regulate the post-transcriptional processing of RNAs, thus affecting the expression of RNAs. HnRNP L can directly target the immediate downstream 5\u0026rsquo; splice-sites of its binding sequence, and subsequently regulate a further downstream exon 5\u0026rsquo; splice-sites selection [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. HnRNP A1 can bind to the stem loop of pri-miRNA-18a and modify the secondary structure of this RNA, thereby generating a more favorable cleavage site for Drosha [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. During the process of influenza A virus (IAV) infection, host hnRNP K binds to virus mRNA and promotes U1 snRNP recruitment, resulting in mRNA mis-splicing to prevent IAV replication [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In the past decades, some hnRNPs are found to be involved in the RNA splicing. However, the relationship between hnRNP-mediated RNA splicing and apoptosis of cancer stem cells has not been explored. In this study, based on the analysis of 18 human hnRNPs, it was found that hnRNP A2B1, upregulated in melanoma stem cells, could suppress apoptosis of melanoma stem cells via post-transcriptional regulation. HnRNP A2B1 takes effects on mRNA stability, mRNA transport, mRNA alternative splicing, cellular senescence and telomere stability by binding single-stranded DNA [\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. However, the function of hnRNP A2B1 in cancer stem cells remains unclear. As reported, hnRNP A2B1 is required for the alternative splicing of apoptosis-related genes, such as the tumor suppressor BIN1 and the anti-apoptotic gene CFLAR (c-FLIP) [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In glioblastoma, breast cancer and pancreatic cancer, hnRNP A2B1 acts as an oncogene by regulating the splicing of apoptosis-associated genes to inhibit apoptosis of cancer cells [\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. These results are consistent with our findings. In this context, our findings provided novel insights into the role of hnRNP-mediated RNA splicing in apoptosis of melanoma stem cells.\u003c/p\u003e \u003cp\u003eAs a splicing factor, hnRNPs can bind to RNAs to mediate the mRNAs\u0026rsquo; transcriptional processing, thereby regulating the expression levels of multiple genes [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. It can regulate mRNA localization [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], mRNA stability [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] and mRNA deadenylation [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. At present, however, the mechanism of hnRNP-mediated apoptosis of cancer stem cells remains unknown. In this study, the results showed that hnRNP A2B1 suppressed apoptosis of melanoma stem cells through post-transcriptional regulation of apoptosis-related \u003cem\u003eDAPK1\u003c/em\u003e, \u003cem\u003eSYT7\u003c/em\u003e, \u003cem\u003eRNF128\u003c/em\u003e, \u003cem\u003eEIF3H\u003c/em\u003e, \u003cem\u003eTPPP3\u003c/em\u003e and \u003cem\u003eDOCK2\u003c/em\u003e genes. As reported, TPPP3, DOCK2 and EIF3H act as oncogenes through suppressing apoptosis of cancer cells [\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], while RNF128, DAPK1 and SYT7 function as tumor suppressors via promoting apoptosis of many types of cancer cells [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Our findings revealed that the hnRNP A2B1-mediated splicing triggered the upregulation of TPPP3, DOCK2 and EIF3H, and the downregulation of RNF128, DAPK1 and SYT7 in melanoma stem cells, leading to the suppression of apoptosis of cancer stem cells. It has been found that the hnRNP A2B1-mediated splicing can increase or decrease the expression levels of its target genes by interacting with other factors [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. In this context, our study revealed a novel mechanism of hnRNP A2B1-mediated suppression of apoptosis of melanoma stem cells. HnRNP A2B1 protein might be a target for melanoma therapy. Except for the apoptosis-associated genes, the other genes of our RNA immunoprecipitation assay, which were upregulated in melanoma stem cells, merited to be further investigated. The proteins interacted with hnRNPA2B1 could be characterized in the future work.\u003c/p\u003e "},{"header":"Conclusion","content":" \u003cp\u003eOur findings revealed that the hnRNP A2B1-mediated splicing triggered the upregulation of TPPP3, DOCK2 and EIF3H, and the downregulation of RNF128, DAPK1 and SYT7 in melanoma stem cells, leading to the suppression of apoptosis of cancer stem cells. HnRNP A2B1 protein might be a target for melanoma therapy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAEC: 3-amino-9-ethylcarbazole\u003c/p\u003e\n\u003cp\u003ebFGF: basic fibroblast growth factor\u003c/p\u003e\n\u003cp\u003eBWA: Burrows Wheeler Aligner\u003c/p\u003e\n\u003cp\u003eCSCs: cancer stem cells\u003c/p\u003e\n\u003cp\u003eDAB: diaminobenzidine\u003c/p\u003e\n\u003cp\u003eDAPK1: death-associated protein kinase 1\u003c/p\u003e\n\u003cp\u003eDOCK2: dedicator of cytokinesis 2\u003c/p\u003e\n\u003cp\u003eEDTA: ethylene diamine tetraacetic acid\u003c/p\u003e\n\u003cp\u003eEGF: epidermal growth factor\u003c/p\u003e\n\u003cp\u003eEIF3H: eukaryotic translation initiation factor 3 subunit H\u003c/p\u003e\n\u003cp\u003eGFP: green fluorescence\u0026nbsp;protein\u003c/p\u003e\n\u003cp\u003eGO: gene ontology\u003c/p\u003e\n\u003cp\u003eHEPES: N-2-hydroxyethylpiperazine-N-ethane-sulphonicacid\u003c/p\u003e\n\u003cp\u003ehg38: human reference 38\u003c/p\u003e\n\u003cp\u003ehnRNPs: heterogenous ribonuclear proteins\u003c/p\u003e\n\u003cp\u003eIACUC: Institutional Animal Care and Use Committee\u003c/p\u003e\n\u003cp\u003eIAV: influenza A virus\u003c/p\u003e\n\u003cp\u003eIGV: Integrative Genomics Viewer\u003c/p\u003e\n\u003cp\u003eKEGG: Kyoto encyclopedia of genes and genomes\u003c/p\u003e\n\u003cp\u003emiRNAs: microRNAs\u003c/p\u003e\n\u003cp\u003eNCBI: National Center for Biotechnology Information\u003c/p\u003e\n\u003cp\u003ePBS: phosphate buffer saline\u003c/p\u003e\n\u003cp\u003epre-mRNAs: precursor mRNAs\u003c/p\u003e\n\u003cp\u003ePVDF: polyvinylidene difluoride\u003c/p\u003e\n\u003cp\u003eRBPs: RNA-binding proteins\u003c/p\u003e\n\u003cp\u003eRIP: RNA immunoprecipitation\u003c/p\u003e\n\u003cp\u003eRNAi: RNA interference\u003c/p\u003e\n\u003cp\u003eRNF128: ring finger protein 128\u003c/p\u003e\n\u003cp\u003eRNP: ribonucleoprotein\u003c/p\u003e\n\u003cp\u003eSDS: sodium dodecyl sulfate\u003c/p\u003e\n\u003cp\u003eshRNA: short hairpin RNA\u003c/p\u003e\n\u003cp\u003esnRNP: small nuclear RNP\u003c/p\u003e\n\u003cp\u003eSYT7: synaptotagmin 7\u003c/p\u003e\n\u003cp\u003eTBS: triethanolamine-buffered saline solution\u003c/p\u003e\n\u003cp\u003eTPPP3: tubulin polymerization promoting protein family member 3\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures conducted on mice in this study were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC). The animal study was reviewed and approved by the Institutional Animal Care and Use Committees of Zhejiang University (Approval No.14843)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the National Key Research and Development Program of China (2018YFC0310703) and China Ocean Mineral Resources R \u0026amp; D Association (DY135-B-04).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXZ conceived of the study conception and designed the work. MC and HW performed all experiments, interpreted data and drafted the work. MC and XZ wrote the manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePlaks V, Kong N, Werb Z. The cancer stem cell niche: how essential is the niche in regulating stemness of tumor cells. Cell stem cell. 2015;16(3):225\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVlashi E, Pajonk F. Cancer stem cells, cancer cell plasticity and radiation therapy. Semin Cancer Biology. 2015;31:28\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrabhu VV, Lulla AR, Madhukar NS, et al. Cancer stem cell-related gene expression as a potential biomarker of response for first-in-class imipridone ONC201 in solid tumors. 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Regulatory expansion in mammals of multivalent hnRNP assemblies that globally control alternative splicing. Cell. 2017;170(2):324\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLoh TJ, Choi N, Moon H, et al. Suppression of 5' splice-sites through multiple exonic motifs by hnRNP L. BBA-Gene Regul Mech. 2017;1860(3):363\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRatnadiwakara M, Mohenska M, \u0026Auml;nk\u0026ouml; ML. Splicing factors as regulators of miRNA biogenesis-links to human disease. Semin Cell Dev Biol. 2018;79:113\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThompson MG, Mu\u0026ntilde;oz-Moreno R, Bhat P, et al. Co-regulatory activity of hnRNP K and NS1-BP in influenza and human mRNA splicing. Nat Commun. 2018;9(1):2407.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGeissler R, Grimson AA. Position-specific 3'UTR sequence that accelerates mRNA decay. RNA Biol. 2016;13(11):1075\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTauler J, Mulshine JL. Lung cancer and inflammation: interaction of chemokines and hnRNPs. Curr Opin Pharmacol. 2009;9(4):384\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVillarroya-Beltri C, Guti\u0026eacute;rrez-V\u0026aacute;zquez C, S\u0026aacute;nchez-Cabo F, et al. Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs. Nat Commun. 2013;4:2980.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGolan-Gerstl R, Cohen M, Shilo A, et al. Splicing Factor hnRNP A2/B1 Regulates Tumor Suppressor Gene Splicing and Is an Oncogenic Driver in Glioblastoma. Cancer Res. 2011;71(13):4464\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDai S, Zhang J, Huang S, et al. 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J Cancer. 2017;8(10):1750\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen YC, Chan JY, Chiu YL, et al. Grail as a molecular determinant for the functions of the tumor suppressor p53 in tumorigenesis. Cell Death Differ. 2013;20(5):732\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSteinmann S, Kunze P, Hampel C. DAPK1 loss triggers tumor invasion in colorectal tumor cells. Cell Death Dis. 2019;10(12):1\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen ZY, Cai L, Zhu J, et al. Fyn requires HnRNPA2B1 and Sam68 to synergistically regulate apoptosis in pancreatic cancer. Carcinogenesis. 2011;32(10):1419\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang S, Yu B, Qian D, et al. Human cytomegalovirus ie2 affects the migration of glioblastoma by mediating the different splicing patterns of RON through hnRNP A2B1. Neuroreport. 2019;30(12):805\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Supplementary Tables","content":"\u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 15px; line-height: 200%; color: black; font-family: Calibri, sans-serif;\"\u003eTable S1. The primer sequences used for quantitative real-time PCR.\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"margin-left:5.4pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 134.45pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0in 5.4pt;height: 9.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eGene\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 280.05pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 9.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003ePrimer\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 134.45pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;height: 22.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003ehnRNP A2B1\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 22.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-GGCAGCATCAACCTCAGCCAT-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-ACTGTTCCTTTGGAGAGG-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 134.45pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003ehnRNP C\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-TGTTTCATAGCATGCGGCAC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-TGGGCTCCA AGGGTAGTTCA-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 134.45pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003ehnRNP D\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-GCGTGGGTTCTGCTTTATTACC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-TT GCTGATATTGTTCCTTCGACA-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 134.45pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003ehnRNP E1\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-CCACGTAACGAGCCCAACT -3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-TTCCGTGCATAAGAAGCCGA-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 134.45pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003ehnRNP E2\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-ACTGGAAGAGGAC ATAAGCA-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-CCAATGAGAGA-GCCACAC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 134.45pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003ehnRNP F\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-CTGCTCTGT TGAGGACGTG-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-CCTGCCCTCTCTAGTGTAGATG-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 134.45pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003ehnRNP G\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-A CATGAATGGAAAGCTCCTGT-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-CGTGGTGGTGGTGCATAATC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 134.45pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003ehnRNP H1\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-AAGGAAAGAATAGGGCACA-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-CTCCACCATAAGCACC AC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 134.45pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003ehnRNP H2\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-AGTGATGCGCTTCTTCTCTGA-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-TTCGGACCTG TATGCTTCAAC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 134.45pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003ehnRNP I\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-CCACGTAACGAGCCCAACT-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-TTCCG TGCATAAGAAGCCGA-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 134.45pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003ehnRNP K\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-CGCTCGTTTTCTGTCTAGCTC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-ACGGGCACACCAATCAGTTA-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 134.45pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003ehnRNP L\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-TACGCAGCCGACAACCAA ATA-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-CTCCGGGAGTCATCCGAGT-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 134.45pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eTPPP3\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-GTCTGTCACCTCTT CATGCA-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-CAGGAGAGCAGAAATCACAC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 134.45pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eDOCK2\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-CTTGGAGG TCCTCAGCTGTC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-GTCTGAGCTGGTCTGGAAGG-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 134.45pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eEIF3H\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-GTG CTTTTGGGTCTGGTTGT-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-ATA-CCAGCCCACGTGAAGAT-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 134.45pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eRNF128\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;- GGGAATT-GAGGTGGATGTTG-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-GTGGCTCATCTGCTCCTTGT-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 134.45pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eDAPK1\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-GATAGAAATGTCCCCAAACCTCG-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-TCTTCTTTGGATCCT TGACCAGAA-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 134.45pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eSYT7\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-ACTC-CATCATCGTGAACATCATC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-TCGA AGGCGAAGGACTCATTG-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 134.45pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eMT-CO1\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-GAGCTGCTGTT-CGGTGTCC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-TGCCAGTGGTAGAGATGGTTG-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 134.45pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eGAPDH\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-GGTATCGTGGAAG GACT CATGAC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 280.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;ATGCCAGTGAGCTTCCCGTTCAG-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eTable S2. The probes used for Northern blot.\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"margin-left:5.4pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113.15pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0in 5.4pt;height: 5.85pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eProbe\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 301.35pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 5.85pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eSequence\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113.15pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;height: 25.3pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003ehnRNP A2B1\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 301.35pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 25.3pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-CTTCCACTCCTAGAACTCTGAACTTCCTGC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113.15pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eTPPP3\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 301.35pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-GCATGGCAGCGAGCACAGACATGG-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113.15pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eEIF3H\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 301.35pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-GGAAATGATGCGGAGCCTTCGCCAT-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113.15pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eDAPK1\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 301.35pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-CAACATCATGCAAAGTGAAACAGT-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113.15pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eSYT7\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 301.35pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-ATTGATAGCCTTCTTCTCACTGCGC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113.15pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eDOCK2\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 301.35pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-GCTTCTCTCAGGAACCTTACCCAAG-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113.15pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eRNF128\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 301.35pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-GCAATAGAGACATGTGAATGTGGCG-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eTable S3. The primers used for semi-quantitative RT-PCR.\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"margin-left:5.4pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 1.65in;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0in 5.4pt;height: 9.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eIntron\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 307.3pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 9.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003ePrimer sequence\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 1.65in;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;height: 22.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eTPPP3\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 307.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 22.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-AACCCTCTTCCACCAAAAAC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 307.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-AAGCACTGGGCGGAT-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 1.65in;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eDOCK2\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 307.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-GTGGACAAATGGGTGAAC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 307.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-CGCATCTCTCATCTCATCC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 1.65in;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eEIF3H\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 307.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-AACTCCCTTTACCTTACTAG-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 307.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-ATTTCTCACTGTCTTCTGCT-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 1.65in;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eRNF128\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 307.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-CAGAGGTTATGTTGCTAG-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 307.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-TAAATCACAACAGCATCAGC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 1.65in;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eDAPK1\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 307.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-AACTCCCTTTACCTTACTAG-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 307.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-ATTTCTCACTGTCTTCTGCT-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 1.65in;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eSYT7\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 307.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-CAAACTACCCACCACGCACT-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 307.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-CTAAGACAAAGTGAGGCAGC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 1.65in;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-style: italic;\"\u003e\u003cspan style=\"line-height: 200%; color: black; font-style: normal;\"\u003e\u0026beta;-tubulin\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 307.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-GCCAGATGCCAAGTGACAAG-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 307.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-TAGTATTCCTCTCCTTCTTC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eTable S4. The siRNA sequences used for gene silencing.\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"margin-left:5.4pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 140.1pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0in 5.4pt;height: 7.8pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003esiRNA\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 274.4pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 7.8pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003esequence\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 140.1pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;height: 23.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003ehnRNP A2B1-siRNA\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 274.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 23.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-AGGAACAGUUCCGUAAGCUCUUUAU-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 140.1pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;height: 11.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003ehnRNP A2B1-siRNA-2\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 274.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 11.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-CGUGCUGTUGCUUGUGUGG-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 140.1pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eTPPP3-siRNA\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 274.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-CCGGGCCAAUGUGGGCGUCACUAAA-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 140.1pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eDOCK2-siRNA\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 274.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-AGGAAGUGACAGUUGAGAA-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 140.1pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eEIF3H-siRNA\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 274.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-GCAACTCTTGGAAGAAATATACTCG-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 140.1pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eRNF128-siRNA\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 274.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-GAGGCAAUUAAAGGCAGAU-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 140.1pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eDAPK1-siRNA\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 274.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-CCGGCCACGUCGAUACCUUG-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 140.1pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eSYT7-siRNA\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 274.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-UCACCGUGAAGAUCAUGAA-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 140.1pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003esiRNA-scrambled\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 274.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-CCGGGCGCGAUAGCGCUAAUA-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eTable S5. The primer sequences used for gene overexpression.\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"margin-left:5.4pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 125.9pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0in 5.4pt;height: 20.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eGene\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 300.2pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 20.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003ePrimer sequence\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 125.9pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;height: 16.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003ehnRNP A2B1 primer 1\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 300.2pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 16.2pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-GCGGAATTCATGGAGAAAACTTTAGAAAC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 125.9pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003ehnRNP A2B1 primer 2\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 300.2pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-ATACTCGAGTCAGTATCGGCTCCTCCCAC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 125.9pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003ehnRNP A2B1 primer 3\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 300.2pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-GGAACAGTTCCGTAAGCACTTTATTGGTGG-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 125.9pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003ehnRNP A2B1 primer 4\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 300.2pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 15pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-CCACCAATAAAGTGCTTACGGAACTGTTCC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 125.9pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;height: 22.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eTPPP3\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 300.2pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 22.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-ATAGAATTCATGGCAGCGAGCACAGACAT-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 300.2pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-GCGCTCGAGTCACTTCTTCACCTTGGCAT-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 125.9pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eDOCK2\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 300.2pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-ATAGATATCATGGCCCCCTGGCGCAAAGC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 300.2pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-TATCTCGAGTCACAGGTCCGTGGACAGCG-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 125.9pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eEIF3H\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 300.2pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-ATAGAATTCATGGCGTCCCGCAAGGAAGG-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 300.2pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-GCGCTCGAGTTAGTTGTTGTATTCTTGAA-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 125.9pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003eRNF128\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 300.2pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; color: black;\"\u003e5\u0026rsquo;-ATAGAATTCATGGGGCCGCCGCCTGGGGC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 300.2pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 200%; 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color: black;\"\u003e5\u0026rsquo;-TATCTCGAGTCAGGCCTTCAGCTGGTGCC-3\u0026rsquo;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Times New Roman\",serif;line-height:200%;'\u003e\u003cspan style=\"font-size: 15px; line-height: 200%; color: black; font-family: Calibri, sans-serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":" Melanoma, cancer stem cells, hnRNP, apoptosis, tumorigenesis","lastPublishedDoi":"10.21203/rs.3.rs-92870/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-92870/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Cancer stem cells play essential roles in tumorigenesis, thus being the important targets for tumor therapy. The hnRNP family proteins, the important splicing factors, are found to be associated with tumor progression. However, the influence of hnRNPs on cancer stem cells has not been extensively explored.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Quantitative real-time PCR and Western blot were used to examine the gene expression level. RNA immunoprecipitation assay and RNA sequencing were conducted to identify the RNAs interacted with hnRNP A2B1 on a genome-wide scale. The in vivo assays were performed in nude mice.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e In this study, the results showed that hnRNP A2B1 of 19 hnRNPs was significantly upregulated in melanoma stem cells compared with non-stem cells, suggesting the important role of hnRNP A2B1 in cancer stem cells. The hnRNP A2B1 silencing triggered the cell cycle arrest in G2 phase, leading to apoptosis of melanoma stem cells. The results revealed that hnRNP A2B1 could bind to the precursor mRNAs of pro-apoptosis genes (\u003cem\u003eDAPK1, SYT7\u003c/em\u003e and \u003cem\u003eRNF128\u003c/em\u003e) and anti-apoptosis genes (\u003cem\u003eEIF3H, TPPP3\u003c/em\u003e and \u003cem\u003eDOCK2\u003c/em\u003e) to regulate the splicing of these 6 genes, thus promoting the expressions of anti-apoptosis genes and suppressing the expressions of pro-apoptosis genes. The \u003cem\u003ein vivo\u003c/em\u003e data indicated that hnRNP A2B1 was required for tumorigenesis of melanoma stem cells \u003cem\u003ein vivo\u003c/em\u003e by affecting the splicing of \u003cem\u003eTPPP3\u003c/em\u003e, \u003cem\u003eDOCK2\u003c/em\u003e, \u003cem\u003eEIF3H\u003c/em\u003e, \u003cem\u003eRNF128\u003c/em\u003e, \u003cem\u003eDAPK1\u003c/em\u003e and \u003cem\u003eSYT7\u003c/em\u003e, thus suppressing apoptosis of melanoma stem cells.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eHnRNP A2B1 was required for tumorigenesis of melanoma stem cells. Therefore our findings presented novel molecular insights into the roles of hnRNPs in cancer stem cells.\u003c/p\u003e","manuscriptTitle":"Requirement of splicing factor hnRNP A2B1 of hnRNPs for tumorigenesis of melanoma stem cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-10-20 22:00:52","doi":"10.21203/rs.3.rs-92870/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2020-11-06T00:00:00+00:00","index":3,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"decision","content":"Major Revision","date":"2020-11-06T00:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-10-27T12:00:00+00:00","index":4,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-10-25T12:00:00+00:00","index":3,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-10-22T12:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorInvitedReview","content":"","date":"2020-10-20T12:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2020-10-17T12:00:00+00:00","index":1,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-10-17T12:00:00+00:00","index":2,"fulltext":""},{"type":"editorAssigned","content":"","date":"2020-10-16T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-10-16T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-10-15T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-10-14T12:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2020-10-13T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4132fe29-42ad-43ae-82d2-d76c59a7c9c6","owner":[],"postedDate":"October 20th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":836664,"name":"Stem Cell \u0026 Developmental Cell Biology"}],"tags":[],"updatedAt":"2021-01-31T15:02:41+00:00","versionOfRecord":{"articleIdentity":"rs-92870","link":"https://doi.org/10.1186/s13287-020-02124-5","journal":{"identity":"stem-cell-research-and-therapy","isVorOnly":false,"title":"Stem Cell Research \u0026 Therapy"},"publishedOn":"2021-01-28 15:02:08","publishedOnDateReadable":"January 28th, 2021"},"versionCreatedAt":"2020-10-20 22:00:52","video":"","vorDoi":"10.1186/s13287-020-02124-5","vorDoiUrl":"https://doi.org/10.1186/s13287-020-02124-5","workflowStages":[]},"version":"v1","identity":"rs-92870","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-92870","identity":"rs-92870","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}