Splicing Factor 3a Subunit 1 Promotes Colorectal Cancer Growth via Anti-programmed cell death of Syntaxin12.

Splicing Factor 3a Subunit 1 Promotes Colorectal Cancer Growth via Anti-programmed cell death of Syntaxin12. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Splicing Factor 3a Subunit 1 Promotes Colorectal Cancer Growth via Anti-programmed cell death of Syntaxin12. Hiroaki Konishi, Takahiro Sasaki, Aki Sakatani, Hiroki Tanaka, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4714660/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract RNA dysregulation mediated by abnormal RNA binding proteins (RBPs) is associated with tumorigenesis. However, the specific tumorigenic mechanisms of each RBP remained unclear. In this study, we demonstrate that splicing factor 3A1 (SF3A1) interacts to and stabilizes the mRNA of STX12, thereby inhibiting programmed cell death (PCD) in colorectal cancer cells. Downregulation of SF3A1 significantly inhibited cell growth in colorectal cancer cells, with minimal cytotoxicity observed in non-cancerous epithelial cells. We validated the tumor-promoting function of SF3A1 in an HCT116 transplanted mouse model. TUNEL staining and western blotting of PARP revealed SF3A1 inhibits PCD in colorectal cancer cells. A transcriptome analysis, combined with RNA-immunoprecipitation (IP), demonstrated that SF3A1 interact to and stabilized 144 mRNAs. Among these mRNAs, knockdown of STX12 (Syntaxin 12) in colorectal cancer cells inhibited cell growth but had no inhibitory effect on non-cancerous epithelial cells, HCEC-1CT. The mRNA levels of STX12 were significantly reduced upon downregulation of SF3A1, contributing to the inhibition of PCD in colorectal cancer cells. Therefore, SF3A1, which mediates STX12 mRNA stabilization, represents a promising therapeutic target for the treatment of colorectal cancer with fewer side effects. Biological sciences/Cell biology/Cell death/Apoptosis Health sciences/Oncology/Cancer/Gastrointestinal cancer RNA binding protein splicing factor 3A1 syntaxin 12 colorectal cancer programed cell death Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The treatment landscape for colorectal cancer (CRC) has significantly progressed through the development of molecularly targeted drugs, immune checkpoint inhibitors 1)2) , and combination chemotherapy employing classical cytotoxic agents such as 5-FU. Despite these advancements, the mortality rate associated with advanced colorectal cancer remains elevated, underscoring the need for the identification and development of efficacious therapeutic targets 3) . Recently, it has been reported that RNA-binding proteins (RBPs) influence cancer development through several mechanisms, including the stabilization of mRNAs and non-coding RNAs, as well as abnormal splicing in cancer cells. We have demonstrated that heterogeneous ribonucleoprotein (hnRNP) A1 exhibits oncogenic properties, which are inhibited by miR-18a, miR-26a, and miR-584 in colon cancer cells 4)5) . Subsequently, hnRNP A1, hnRNP A2, and polypyrimidine tract-binding protein (PTB), highly induced in colorectal cancer cells 6)7)8)9) , were reported to facilitate the alternative splicing of pyruvate kinase M1 (PKM1) to PKM2, thereby supporting tumor growth 10) . Additionally, RBPs can promote tumorigenesis not only through abnormal expression but also via post-translational modifications such as phosphorylation 11)12)13) . We observed that hnRNP A0 is specifically phosphorylated in cancer cells, and its phosphorylated form selectively binds to and stabilizes oncogenic RAB3GAP1 mRNA, resulting in the inhibition of tumor cell apoptosis and the promotion of colorectal cancer (CRC) development 6) . Furthermore, through a high-throughput screening involving 1198 siRNAs targeting 416 human RBPs in colorectal, esophageal, and pancreatic cancer cells, coupled with comprehensive expression analysis using cDNA arrays, we identified 12 RBPs, including SF3A1, that undergo post-translational modifications in cancer cells, thereby promoting cancer cell growth 14) . SF3A1 is a subunit of the SF3a complex, an integral component of U2 snRNP, playing a crucial role in intron recognition and pre-RNA splicing during the splicing process 15) . Notably, we observed that SF3A1 exerts its growth-promoting effects specifically in cancer cells, despite no discernible alteration in expression levels between tumor and normal cells 14) . Genetic mutations in SF3A1 may play a role in pancreatic cancer development 16) , and a reported correlation exists between SF3A1 gene polymorphisms and colorectal cancer (CRC) risk 17) . However, the specific mRNAs with which SF3A1 interacts in CRC and whether this interaction is directly implicated in tumor growth remain unclear. In this study, our purpose was to elucidate the mechanisms of SF3A1-mediated tumor progression in colorectal cancer cells. Results ・SF3A1 enhances the tumor progression in colorectal cancer cells, but not in non-cancerous cells Our previous study suggested that 12 RBPs (RPS3, RBM22, EIF2S1, DHX8, RBM8A, UPF1, YBX1, SNRPE, SF3A1, U2AF1, SUPT6H, and EIF3G) exhibit tumor-promoting properties through posttranslational modifications in cancer cells 14) . To validate the tumorigenic properties of each molecule in colorectal cancer cells, we conducted the sulforhodamine B (SRB) assay using the colorectal cancer cells, HCT116, and non-cancerous epithelial cells, HCEC-1CT, with downregulation of each RBP (Table 1). In this study, tumor-promoting RBPs were defined as RBPs that, when downregulated by siRNA, completely inhibit the proliferative potential of the cancer cell line HCT116 (resulting in > 100% growth inhibition) and have a slight effect (< 30% inhibition) on the proliferative potential of the non-tumor cell line HCEC-1CT. SF3A1 exhibited the highest promotion of tumor cell growth and the least effect on non-tumor cell growth. (Table 1). Downregulation of SF3A1 resulted in growth suppression in SW480 cells as well as HCT116 cells. No difference in SF3A1 mRNA expression was observed in colorectal cancer cells and non-cancerous epithelial cells (Fig. 1 A, 1 B, the knockdown efficacy was shown in Fig. 1 C). An analysis using GEPIA ( http://gepia.cancer-pku.cn/ ) of 275 colorectal cancer tissue and 349 normal tissue showed that the expression of SF3A1 did not significantly change CRC patients (Supplemental Fig. 1). To explore the in vivo tumor-promoting function of SF3A1, HCT116 cells were subcutaneously transplanted on the left and right sides of the backs of nude mice, and siRNA targeting SF3A1 or Scramble RNA was directly transfected into the transplanted HCT116 cells through local injection. The downregulation of SF3A1 significantly inhibited tumor growth (Fig. 1 D). These results suggest that SF3A1 exerts its growth-promoting effects selectively in cancer cells, despite no difference in SF3A1 expression levels between tumor and non-tumor cells. ・SF3A1 promotes proliferation and inhibits programmed cell death (PCD) of colorectal cancer cells To elucidate the role of SF3A1 in cellular proliferation, immunocytochemistry for Ki-67 was performed in SF3A1-downregulated HCT-116 cells. The number of Ki-67-positive cells significantly decreased with the downregulation of SF3A1 (Fig. 2 A). Subsequently, to investigate whether SF3A1 inhibits programmed cell death, TUNEL staining was conducted. TUNEL-positive cells exhibited a significant increase in SF3A1-downregulated HCT116 cells (Fig. 2 B). Meanwhile, the number of TUNEL and Ki-67 positive cells were not significantly changed in SF3A1-downregulated HCEC-1CT (Supplemental Fig. 2). Western blotting further revealed an elevation in PARP cleavage in SF3A1-downregulated HCT116 cells (Fig. 2 C). These results strongly suggest that SF3A1 promotes the development of colorectal cancer cells by enhancing cell proliferation and inhibiting PCD. ・SF3A1 promotes the progression of colorectal cancer cells through stabilizing the mRNA of Syntaxin12 (STX12). To identify mRNAs directly interacting with SF3A1 in colorectal cancer cells, immunoprecipitation was conducted using an SF3A1 antibody to pull down SF3A1 along with its interacting RNAs from HCT116 cell lysates (Supplemental Fig. 3). Subsequently, transcriptome analysis was performed using RNA extracted from the precipitant after removing proteins and DNAs. A total of 7283 mRNAs were found to interact with SF3A1 in HCT116 cells (Supplemental Table 1). To identify mRNAs whose expressions were altered by the downregulation of SF3A1, transcriptome analysis was carried out in SF3A1-downregulated HCT116 cells, revealing that the expressions of 477 mRNAs significantly decreased upon SF3A1 downregulation (Supplemental Table 2). By integrating the IP-transcriptome and whole transcriptome analysis data, 144 mRNAs were identified as being stabilized through direct interacting with SF3A1 (Fig. 3 A). Among these, we selected the top 10 mRNAs exhibiting the most significant expression changes (Table 2). To determine the association of these 10 mRNAs with tumor promotion in colorectal cancer cells, we generated siRNAs targeting each mRNA. Subsequently, each siRNA was transfected into HCT116 and HCEC-1CT cells, and their effects on tumor growth inhibition were analyzed (Fig. 3 B). The SRB assay revealed that downregulation of Syntaxin12 (STX12) exhibited cytotoxic effects in HCT116 but not in HCEC-1CT cells (Fig. 4 A, the knockdown efficacy was shown in Fig. 4 B). Additionally, RT-PCR demonstrated that STX12 mRNA was reduced by the downregulation of SF3A1 in HCT116, SW480, and HCEC-1CT (Fig. 4 C). These results indicate that SF3A1 significantly contributes to the progression of colon cancer cells by stabilizing STX12 mRNA, whereas STX12 exhibits minimal impact on the growth of non-cancerous cells, specifically HCEC-1CT. ・Syntaxin12 (STX12) inhibits the PCD in colorectal cancer cells Considering that SF3A1 promotes cell proliferation and inhibits PCD in colorectal cancer cells, we conducted immunocytochemistry for Ki-67 and TUNEL staining in STX12-downregulated cells. Contrary to expectations, immunocytochemistry for Ki-67 revealed that the number of proliferative cells remained unchanged upon the downregulation of STX12 in HCT116 cells (Fig. 5 A). In contrast, TUNEL staining demonstrated a significant increase in DNA fragmented cells in the STX12-downregulated HCT116 cells (Fig. 5 B). The number of TUNEL and Ki-67 positive cells were not changed in STX12 downregulated HCEC-1CT (Supplemental Fig. 4). Western blotting further indicated the induction of PARP cleavage in the STX12-downregulated HCT116 cells (Fig. 5 C). These findings suggest that SF3A1 stabilizes STX12 mRNA, thereby exerting an anti-PCD, but not a proliferative, effect on colorectal cancer cells. Discussion In this study, we demonstrated that the RNA-binding protein SF3A1 promotes tumor progression in both in vitro and in vivo models of colorectal cancer. Furthermore, we elucidated the tumor-promoting roles of SF3A1, mediating its direct interaction with STX12 mRNA, thereby facilitating its stabilization and suppressing PCD within colon cancer cells. TUNEL staining revealed that SF3A1 possesses anti-PCD function against colorectal cancer cells. Our pull-down assay demonstrated that SF3A1 interacts to STX12 mRNA, and transcriptome analysis in SF3A1-downregulated cells revealed that the mRNA expression of STX12 was regulated by SF3A1. Furthermore, downregulation of STX12 was associated with an augmentation of PCD in colon cancer cells. These results clearly indicate that SF3A1 inhibits PCD in colon cancer cells by increasing the expression of STX12 mRNA. Previous investigations have indicated the tumor-promoting potential of STX12. Lee et al. demonstrated that STX12 is upregulated via the ROS/STAT3/NFE2L1 pathway, supporting tumor progression in hepatocellular carcinoma cells 18) . STAT3 is a known key molecule involved in the development of colorectal cancer cells 19)20) , and SF3A1 may promote colorectal cancer development by stabilizing STAT3-STX12-mediated antiapoptotic signaling. Additionally, STX12 suppresses the expression of miR-148a 21) , which represses NFkB signaling, by binding to the 3'-UTR of TLR4 mRNA. Therefore, the STX12-miR-148a pathway also associates with tumor-promoting functions in colorectal cancer cells. Interestingly, bioinformatic analysis showed that the expression of STX12 was not significantly changed CRC patients (Supplemental Fig. 5). Malik et al. showed that STX12 is phosphorylated by SGK3 22) , which is known as a downstream mediator of phosphatidylinositol 3-kinase (PI3K) oncogenic signaling 23) , indicating that post-transcriptional modification, as well as the expression of STX12, plays a crucial role in colorectal cancer progression. Our Ki67 staining revealed that SF3A1 possesses proliferative and anti-PCD functions against colorectal cancer cells, whereas STX12 exhibits only anti-PCD function, with less effect on cell proliferative capacity. In relation to the SF3A1-induced proliferation of colon cancer cells, our pull-down assay combined with transcriptome analysis identified various mRNAs, other than STX12, that interacted with SF3A1 and have cell proliferative properties, such as RASSF6 24) and SULF2 25) . This suggests that SF3A1 may promote cancer cell proliferation through stabilizing these mRNAs. Further analysis of the functions of these molecules, in which SF3A1 interacts to and stabilizes their mRNAs, is needed to identify the responsible molecules mediating the cell proliferation effect of SF3A1 in cancer cells. The SRB assay, TUNEL staining and Ki-67 staining revealed that the downregulation of SF3A1 significantly suppressed colorectal cancer cells while showing no impact on non-tumor cells. Notably, there was no significant expression change of SF3A1 between colorectal cancer and non-tumor cells. These observations suggest that SF3A1 undergoes specific post-translational modifications in cancer cells, thereby exerting oncogenic properties, including anti-PCD and proliferative functions. Phosphorylation is a well-known major type of post-translational modification that activates protein function. In our previous study, we demonstrated that hnRNP A0, an RNA-binding protein, stabilized oncogenic RAB3GAP1 mRNA via MAPKAP2 and supported tumor progression, specifically when Ser84 in hnRNP A0 was phosphorylated 13) . SF3A1 possesses 23 serine/threonine residues, and the phosphorylation of these sites might be associated with SF3A1-induced stabilization of target mRNAs in colon cancer cells ( https://www.phosphosite.org/proteinAction.action?id=4183&showAllSites=true ). Understanding the mechanisms of post-translational modification of SF3A1, including phosphorylation, ubiquitination, glycosylation, acetylation, and others, is expected to facilitate the development of therapeutic targets associated with SF3A1 protein. Several RNA-binding proteins (RBPs), including RBM22 26) and U2AF1 27) , along with SF3A1, have been reported to exert tumor-specific promotion, suggesting that these RBPs may also undergo post-translational modifications in cancer cells. However, the detailed target RNAs of these RBPs remain unknown. Our previous investigations have identified numerous RBPs upregulated in colorectal cancer cells, such as hnRNP A1, hnRNP H1, and RBMXL2. However, the main targets of mRNAs differ, and each RBP regulates distinct mechanisms, such as the cell cycle and sphingolipid metabolism in cancer cells 4)5)28)29) . A detailed analysis of the mechanisms by which each RBP contributes to tumor-promoting effects is expected to facilitate the development of cancer therapy strategies based on changes in RBP expression and post-translational modifications. Despite comparable knockdown efficiencies, the SRB assay also indicated that the inhibitory effect on cell proliferation observed in SW480 by SF3A1 suppression was less than in HCT116. This suggests the variation in the post-translational modification status of SF3A1 even among colon cancer cells. Studies aimed at elucidating the character of cancers that alter the status of post-translational modifications of SF3A1 will enable us to predict the efficacy of SF3A1-targeted cancer therapies. In this study, we have elucidated the functional alterations of SF3A1, an RNA-binding protein, in colorectal cancer cells, despite its comparable expression levels between tumor and non-tumor cells. This observation underscores its involvement in tumor growth. Furthermore, our findings reveal that SF3A1 plays a pivotal role in promoting tumor growth by exerting an anti-PCD effect through its direct interaction with STX12. Notably, inhibition of SF3A1 results in a significant suppression of tumor growth specifically in colorectal cancer cells, without affecting non-tumor cells. This study represents to identify the SF3A1-STX12 mRNA pathway as a potential therapeutic target for colorectal cancer. Methods Cell culture Human CRC cell lines (HCT116 and SW480) (American Type Culture Collection [ATCC]) were cultured in McCoy's 5A medium (HCT116) and Roswell Park Memorial Institute 1640 medium (SW480). The media were supplemented with 10% (vol/vol) fetal bovine serum (FBS) and 50 U/ml penicillin, and cells were maintained at 5% CO2 and 37°C under humidified conditions. Human colon epithelial cells (HCEC-1CT) (Summit Pharmaceuticals International Corporation) were cultured in ColoUp medium (DMEM/Medium 199 Earle’s, 4 + 1; Biochrom Cat# F0435 and Cat# FG0615). The medium included 4 mM GlutaMAXTM-1 (100×, Gibco, Cat# 35050-038), 2% cosmic calf serum (Hyclone, Cat# SH30087), 20 ng/ml EGF (Sigma Aldrich, Cat# E9644), 10 µg/ml Insulin (Sigma Aldrich, Cat# I9278), 2 µg/ml Apo-Transferrin (Sigma Aldrich, Cat# T2036), 5 nM sodium-selenite (Sigma Aldrich, Cat# S5261), and 1 µg/ml hydrocortisone (Sigma Aldrich, Cat# H0396). The cells were maintained at 5% CO2 and 37°C under humidified conditions. siRNA and Transfection 12 siRNAs targeting RBPs 14) and a total of 18 siRNAs targeting 10 mRNAs, except for EIF4EBP3 (Table 2), were selected from the Silencer Select siRNA Libraries (Thermo Fisher Scientific) (Supplemental table 3 ). EIF4EBP3 siRNA was generated by annealing two synthetic RNAs as follows: sense, 5'-UGGAUUAGAUGUCCAUUUCAA-3' and antisense, 5'-GAAAUGGACAUCUAAUCCAGU-3' (Hokkaido System Science Co., Ltd.). A negative control, consisting of a scrambled RNA sequence, was prepared by annealing two synthetic RNAs as follows: sense, 5'-UACGUACUAUCGCGCGGAU-3' and antisense, 5'-AUCCGCGCGAUAGUACGUA-3' (Hokkaido System Science Co., Ltd.). Transfection was carried out in triplicate using Lipofectamine RNAiMAX (Thermo Fisher Scientific) following the manufacturer's instructions for reverse transfection methods. SRB assays The cells were seeded onto 96-well microplates at a density of 0.75 × 10 4 cells per well. Subsequently, the cells were fixed using a solution of 5% trichloroacetic acid (TCA) at 4 ℃ for one hour. Following fixation, the plates were rinsed four times with distilled water, dehydrated at room temperature, and then stained with 100 µL/well of 0.057% (wt/vol) sulforhodamine B (SRB) powder in 0.1% acetic acid. After staining, the microplates were washed four times with 0.1% acetic acid and re-dehydrated at room temperature. The cells were lysed in a 10 mM Tris-buffered environment, and the optical density (OD) was measured at a wavelength of 510 nm. Real-time PCR Total RNA was isolated utilizing the RNeasy Mini Kit (Qiagen) following the manufacturer's instructions. cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). The Ct values were monitored across a spectrum using the Applied Biosystems 7300 Real-Time PCR system, employing Taqman gene expression assays for SF3A1 (Hs01066327) and STX12 (Hs00295291) in duplicate. The mRNA expression of 18S rRNA served as the normalization reference for each sample. Xenografts The dorsal lateral region of male BALB/c nude mice, aged 6 to 8 weeks, was subjected to a subcutaneous injection of HCT116 cells (2 × 10 6 cells). After the tumor tissue reached 4–5 mm, SF3A1 siRNA or scramble siRNA was administered daily using the GENOMONE-Si transfection kit (Ishihara Sangyo, Co, Ltd.) through local injection into the transplanted tumor. Tumor diameter was measured daily before transfection. The tumor sizes were calculated from digital caliper raw data using the following formula: Volume = (major tumor diameter) × (minor tumor diameter). Immunocytochemistry HCT116 cells were cultured on glass chamber slides and subsequently fixed using a 4% paraformaldehyde solution at 4°C. The cells underwent washing with PBS, followed by permeabilization using a 0.1% Triton X-100 solution and subsequent blocking with 3% BSA in PBS. Subsequently, the slides were incubated overnight at 4°C with primary antibodies (Ki-67 [Novus, NB500-170]), followed by PBS washing and subsequent incubation with Alexa 488-conjugated secondary antibodies (ThermoFisher Scientific) for 1 hour at room temperature. The nuclei were counterstained with 4’,6-Diamidine-2’-phenylindole dihydrochloride (Sigma Aldrich). TUNEL staining HCT116 cells were cultured on glass chamber slides and subsequently fixed with a 4% paraformaldehyde solution at 4°C. The cells underwent PBS washing, followed by staining using an In Situ Cell Death Detection Kit with TMR red (Roche Diagnostic) according to the manufacturer's instructions. The cells were mounted with an anti-fade mounting medium, and immunofluorescence was visualized using a fluorescence microscope (KEYENCE Corporation). RNA-immunoprecipitation HCT116 cells were solubilized in NP-40 cell lysis buffer (Thermo Fisher Scientific), supplemented with a complete protease inhibitor cocktail (Merck) and RNasin (Promega Corporation). The lysates were centrifuged at 20,000×g for 5 minutes to remove cellular debris. RNAs forming a complex with SF3A1 were selectively pulled down using either SF3A1 antibody (ThermoFisher Scientific ; A301-602A) or an isotype control antibody, employing a Dynabeads immunoprecipitation kit (VERITAS Corporation). The precipitated RNAs were isolated using phenol–chloroform extraction and purified with a mirVana™ Isolation Kit (ThermoFisher Scientific). Transcriptome analyses RNA libraries were generated using the Ion Total RNA-Seq Kit v2 (ThermoFisher Scientific) following the manufacturer’s instructions. The RNA libraries underwent an emulsion polymerase chain reaction (PCR) process utilizing the Ion OneTouchTM system and the Ion OneTouch 200 Template kit v3 (ThermoFisher Scientific). Template-positive Ion SphereTM particles were enriched and purified, prepared for subsequent sequencing using the Ion OneTouchTM ES system (ThermoFisher Scientific). Following this, the template-positive Ion SphereTM Particles were loaded onto Ion PI™ Chips (ThermoFisher Scientific), and high-throughput sequencing was performed using the Ion Proton™ Semiconductor sequencer (ThermoFisher Scientific). The entire sequencing dataset was aligned to the human reference genome sequence (GRCh37/hg19) using the Torrent Suite software program (ThermoFisher Scientific). After importing the expression data for each sample into the CLC Genomics Workbench software program (CLC bio, Aarhus, Denmark), distinctions among the samples were assessed using an unpaired t-test to determine their statistical significance. Western blotting HCT116 cells were lysed in NP-40 cell lysis buffer (ThermoFisher Scientific) supplemented with cOmplete™ Protease Inhibitor Cocktail (Merck). Following centrifugation at 20,000g for 5 minutes, the lysate was denatured with Laemmli Sample Buffer containing 2-mercaptoethanol at 95℃ for 5 minutes. Equal amounts of protein were loaded onto an SDS–PAGE gel (12.5%), followed by transfer onto a nitrocellulose membrane at 100 V for 60 minutes. The blots were blocked in SuperBlock T-20 (PBS; ThermoFisher Scientific) for 1 hour, then incubated with the primary antibody in SuperBlock T‐20. The primary antibody used, specifically the cleaved poly-ADP-ribose polymerase (PARP) (#9546, Cell Signaling Technology, Inc.), was diluted to 1:1000 in SuperBlock T‐20 (PBS). Subsequently, an overnight incubation with the blots was conducted at 4℃. The blots were washed in 0.05% Tween20‐PBS (T‐PBS) three times for 15 minutes and then incubated in SuperBlock T‐20 (PBS) containing HRP-conjugated secondary antibodies (R&D Systems, Inc.). Following three washes in T-PBS for 15 minutes each, the blots were visualized using the Super Signal West Pico enhanced chemiluminescence system (ThermoFisher Scientific). Actin (612656, BD Transduction Laboratories) protein expression was used for normalizing protein levels. Statistical Analyses Statistical significance was assessed using Student's t-test, with p-values below 0.05 considered indicative of statistical significance. Declarations Ethics Statement Animal Studies: The studies were approved by the use of an opt-out methodology from the Medical Ethics Committee of Asahikawa Medical University (Approval No. R5-059). Conflict of Interest Mikihiro Fujiya and Hiroaki Konishi was funded by Kamui phama, Inc. Funding Information This paper was supported by Grants-in-Aid for Scientific Research, No. 21K07929 (M. Fujiya), 22K15363 (H. Konishi), 21KK0291 (H. Konishi) and 22K08047 (K. Moriichi), Intractable Disease Health and Labour Sciences Research Grants from the Ministry of Health, Labour and Welfare (M. Fujiya). Author Contributions T.S. and H.K., and M.F. provided major input regarding the conceptual development of the studies, wrote the manuscript, and supervised all of the investigations. T.S., H.K, and H.T. performed the biochemical experiments. A.S., K.Y., K.T., K.A., N.U., S.K., K.M., H.T., and T.O. helped to design the studies, interpret the data, and prepare/review the manuscript. All the authors read and approved the final manuscript. Acknowledgments: The authors thank Chikage Yamamura, and Nobue Tamamura for their valuable technical assistance. 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Cancer Lett. 389 , 11–22 (2017). 10.1016/j.canlet.2016.12.033 Epub 2016 Dec 30 Rosen, S.D., Lemjabbar-Alaoui, H.: Sulf-2: an extracellular modulator of cell signaling and a cancer target candidate. Expert Opin. Ther. Targets. 14 (9), 935–949 (2010). 10.1517/14728222.2010.504718 Soubise, B., Jiang, Y., Douet-Guilbert, N., Troadec, M.B.: RBM22, a Key Player of Pre-mRNA Splicing and Gene Expression Regulation, Is Altered in Cancer. Cancers (Basel). 14 (3), 643 (2022). 10.3390/cancers14030643 Palangat, M., Anastasakis, D.G., Fei, D.L., et al.: The splicing factor U2AF1 contributes to cancer progression through a noncanonical role in translation regulation. Genes Dev. 33 (9–10), 482–497 (2019). 10.1101/gad.319590.118 Epub 2019 Mar 6 Takahashi, K., Fujiya, M., Konishi, H., et al.: Heterogenous Nuclear Ribonucleoprotein H1 Promotes Colorectal Cancer Progression through the Stabilization of mRNA of Sphingosine-1-Phosphate Lyase 1. Int. J. Mol. Sci. 21 (12), 4514 (2020). 10.3390/ijms21124514 PMID: 32630435; PMCID: PMC7350029 Murakami, Y., Konishi, H., Fujiya, M., et al.: Testis-specific hnRNP is expressed in colorectal cancer cells and accelerates cell growth mediating ZDHHC11 mRNA stabilization. Cancer Med. 11 (19), 3643–3656 (2022). 10.1002/cam4.4738 Epub 2022 Apr 5. PMID: 35384384; PMCID: PMC9554453 Tables Table.1 SF3A1 has the least effect on non-tumor cells but the greatest effect on colon cancer cells. HCEC-ICT HCT116 RPS3 56% 93% RBM22 35% 110% EIF2S1 60% 91% DHX8 21% 88% RBM8A 71% 120% UPF1 46% 71% YBX1 39% 66% SNRPE 45% 112% SF3A1 29% 111% U2AF1 24% 80% SUPT6H 50% 105% EIF3G 40% 97% The inhibition rate of the SRB assay using the colorectal cancer cells, HCT116, and non-cancerous epithelial cells, HCEC-1CT, with downregulation of each RBP. The inhibition rate (%) = [1 – (OD510 nm at day 3 of siRNA of each RBP- OD510 nm at day 1 of siRNA of each RBP) / (OD510 nm at day 3 of Scrambled RNA- OD510 nm at day 1 of Scrambled RNA)] × 100. The cut-off for HCEC-1CT was defined as 100%. Table.2 The top 10 mRNAs with the largest changes. IP-Teranscriptome Analysis (SF3A1/IgG) Transcriptome Analysis (SF3A1/scramble) Fold Change p-Value Fold Change p-Value MGC32805 30.48 0.0008 -9.19 0.013 DRICH1 ------ 0.0246 -8.30 0.011 DNAJC5G 7.15 0.0036 -6.74 0.008 SLCO1B3 61.46 0.0137 -5.62 0.012 GEMIN2 3.35 0.0157 -5.51 0.005 EIF4EBP3 6.29 0.0228 -5.50 0.003 MRPS31P 21.88 0.0090 -5.00 0.006 STX12 4.63 0.0069 -4.90 0.007 VIM-AS1 7.42 0.0139 -4.90 0.023 LUCAT1 24.73 0.0011 -4.59 0.012 Additional Declarations Yes there is potential Competing Interest. Mikihiro Fujiya and Hiroaki Konishi was funded by Kamui phama, Inc. Supplementary Files Supplementalfigure1.pptx Supplementalfigure2.pptx Supplementalfigure3.pptx Supplementalfigure4.pptx Supplementalfigure5.pptx Supplementaltable1.xlsx Supplementaltable2.xlsx Supplementaltable3.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4714660","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":342061089,"identity":"bfdd004f-943b-497b-8323-9fe638a37e6d","order_by":0,"name":"Hiroaki 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University","correspondingAuthor":false,"prefix":"","firstName":"Takahiro","middleName":"","lastName":"Sasaki","suffix":""},{"id":342061091,"identity":"b3013023-9719-45cf-80c7-bab88acaa71d","order_by":2,"name":"Aki Sakatani","email":"","orcid":"","institution":"Asahikawa Medical University","correspondingAuthor":false,"prefix":"","firstName":"Aki","middleName":"","lastName":"Sakatani","suffix":""},{"id":342061092,"identity":"0c326311-dbc7-4745-9e12-fb60fe0cb4b9","order_by":3,"name":"Hiroki Tanaka","email":"","orcid":"","institution":"Asahikawa Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hiroki","middleName":"","lastName":"Tanaka","suffix":""},{"id":342061093,"identity":"30ec1bba-9db9-46e8-8120-b1c3323ef70b","order_by":4,"name":"Koji Yamamoto","email":"","orcid":"","institution":"Asahikawa Medical 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University","correspondingAuthor":false,"prefix":"","firstName":"Hiroki","middleName":"","lastName":"Tanabe","suffix":""},{"id":342061100,"identity":"c79e3035-7b77-477e-a996-a47f401f74a5","order_by":11,"name":"Toshikatsu Okumura","email":"","orcid":"","institution":"Asahikawa Medical University","correspondingAuthor":false,"prefix":"","firstName":"Toshikatsu","middleName":"","lastName":"Okumura","suffix":""},{"id":342061101,"identity":"3f9ca8c4-81c1-4742-8523-fe99f4995638","order_by":12,"name":"Mikihiro Fujiya","email":"","orcid":"","institution":"Asahikawa Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mikihiro","middleName":"","lastName":"Fujiya","suffix":""}],"badges":[],"createdAt":"2024-07-10 00:20:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4714660/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4714660/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":64625300,"identity":"efbbda62-0ec9-4d7d-a2a2-03c3f416fc9c","added_by":"auto","created_at":"2024-09-16 17:54:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":954188,"visible":true,"origin":"","legend":"\u003cp\u003eSF3A1 enhances the tumor progression in colorectal cancer cells, but not in non-cancerous cells.\u003c/p\u003e\n\u003cp\u003e(A) An SRB assay showed SF3A1 downregulation led the growth suppression in cancer cells (HCT116, SW480), but not non-cancerous epithelial cells (HCEC-1CT) (n=5). (B)RT-PCR showed no difference in SF3A1 mRNA expression levels in HCT116, SW480 and HCEC-1CT cells(n =3) (C)RT-PCR showed the knockdown efficacy of HCT116, SW480 and HCEC-1CT cells transfected with siRNA of SF3A1(n=3). (D) The size of the HCT116 xenograft tumor were assessed on a daily basis. *P \u0026lt; 0.05, **P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4714660/v1/d48030e4f9a91af829ded451.png"},{"id":64625299,"identity":"6e5d8888-9a4b-4f91-95e3-03cc3f877db5","added_by":"auto","created_at":"2024-09-16 17:54:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1899627,"visible":true,"origin":"","legend":"\u003cp\u003eSF3A1 promotes proliferation and inhibits programmed cell death of colorectal cancer cells.\u003c/p\u003e\n\u003cp\u003e(A) Immunocytochemistry showed that the downregulation of SF3A1 resulted in a decrease in Ki-67 positive cells. (B) TUNEL staining showed that the downregulation of SF3A1 resulted in an increase in TUNEL positive cells. (C) Western blots showed enhanced PARP cleavage in SF3A1-downregulated HCT116 cells. *P \u0026lt; 0.05, **P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4714660/v1/0c2fcd7761225acd56a20d68.png"},{"id":64625309,"identity":"131917c3-639c-43c1-bd23-57bca7b954ff","added_by":"auto","created_at":"2024-09-16 17:54:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1071026,"visible":true,"origin":"","legend":"\u003cp\u003eSF3A1 promotes the progression of colorectal cancer cells through stabilizing the mRNA of Syntaxin12 (STX12).\u003c/p\u003e\n\u003cp\u003e(A) SF3A1 was immunoprecipitated from the lysate of HCT116 cells, and then conducted transcriptome analysis using RNA extracted from the precipitant (value of fold change; \u0026gt; 2, p \u0026lt; 0.01). 7283 mRNAs interact with SF3A1. Alterations in mRNA profiles stemming from the downregulation of SF3A1 were examined through transcriptome analysis of HCT116 cells transfected with SF3A1 siRNA compared to IgG (value of fold change; \u0026gt; 2, p \u0026lt; 0.01). 477 mRNAs with significantly altered by the downregulation of SF3A1. We identified 144 mRNAs that were stabilized through direct interacting with SF3A1. (B) 10 siRNA of mRNA was transfected to HCT116 and HCEC-1CT cells, and their tumor growth inhibitory effects were confirmed. SRB assay showed that downregulation of Syntaxin12 (STX12) showed the most cytotoxic function in HCT116 cells, but not in HCEC-1CT cells.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4714660/v1/474ee65456284a38ffb8abcd.png"},{"id":64625305,"identity":"36dad9ce-3b0a-4459-acfd-0b1a05d6fec4","added_by":"auto","created_at":"2024-09-16 17:54:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":423755,"visible":true,"origin":"","legend":"\u003cp\u003eSTX12 enhances the tumor progression in colorectal cancer cells, but not in non-cancerous cells.\u003c/p\u003e\n\u003cp\u003e(A) An SRB assay showed STX12 downregulation led the growth suppression in cancer cells (HCT116, SW480), but not non-cancerous epithelial cells (HCEC-1CT) (n=5). (B)RT-PCR showed the knockdown efficacy of HCT116, SW480 and HCEC-1CT cells transfected with siRNA of STX12(n=3). (C)RT-PCR showed that downregulation of HCT116 with si RNA of SF3A1 reduced STX12. *P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4714660/v1/4d3bd50d8525ece932fc403d.png"},{"id":64625303,"identity":"d6e9d331-cf8c-4254-9420-fe544210adb2","added_by":"auto","created_at":"2024-09-16 17:54:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2394952,"visible":true,"origin":"","legend":"\u003cp\u003eSyntaxin12 (STX12) inhibits the programed cell deth in colorectal cancer cells.\u003c/p\u003e\n\u003cp\u003e(A) Immunocytochemistry showed that the downregulation of STX12 did not result in a decrease in Ki-67 positive cells. (B) TUNEL staining showed that the downregulation of STX12 resulted in an increase in TUNEL positive cells. (C) Western blots showed enhanced PARP cleavage in SF3A1-downregulated HCT116 cells. *P \u0026lt; 0.05, **P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4714660/v1/25f78936f23d61f9ffc2e487.png"},{"id":66310001,"identity":"8d0dec76-de8f-4f17-9256-f55f595ab7b0","added_by":"auto","created_at":"2024-10-10 08:24:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6378918,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4714660/v1/70560d3d-a84e-4eaa-85fd-a68ac8e1a5a5.pdf"},{"id":64625298,"identity":"6d7ffdc2-937d-475c-b032-0dd29450634d","added_by":"auto","created_at":"2024-09-16 17:54:51","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":69681,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalfigure1.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4714660/v1/70b9086f4bcf74bf507a420b.pptx"},{"id":64625310,"identity":"5342c6e9-e829-4092-a9d1-2e94454a7aea","added_by":"auto","created_at":"2024-09-16 17:54:52","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":44209707,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalfigure2.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4714660/v1/ad32348be586b00bb1de895d.pptx"},{"id":64625302,"identity":"21660110-77c7-4ce8-b60b-4c467b7c1005","added_by":"auto","created_at":"2024-09-16 17:54:51","extension":"pptx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":101286,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalfigure3.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4714660/v1/009e0ece2c0b0c9de62a7e3f.pptx"},{"id":64625311,"identity":"23f4cd34-adf8-4dbe-82c8-db84d2633a6a","added_by":"auto","created_at":"2024-09-16 17:54:52","extension":"pptx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":23280672,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalfigure4.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4714660/v1/b6ea9b64dc42f3b3e48c971b.pptx"},{"id":64625306,"identity":"e99b2677-6582-4abd-8b29-72495729b46a","added_by":"auto","created_at":"2024-09-16 17:54:51","extension":"pptx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":67313,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalfigure5.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4714660/v1/7db556ed89ca3d2679e444cd.pptx"},{"id":64625304,"identity":"0f48c6e2-fec7-4d19-ae13-7012a07a3f99","added_by":"auto","created_at":"2024-09-16 17:54:51","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":341551,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaltable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4714660/v1/a73244d85cf38ed68a960b81.xlsx"},{"id":64625307,"identity":"f50de18f-41f1-417b-aa66-7e5fabac8d9b","added_by":"auto","created_at":"2024-09-16 17:54:51","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":35028,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaltable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4714660/v1/3205ccad0dad78610710cc98.xlsx"},{"id":64625585,"identity":"66c3c60f-eec0-4a32-80bb-71ca9f66e63d","added_by":"auto","created_at":"2024-09-16 18:02:51","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":10633,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Supplementaltable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4714660/v1/328c2aac67548b465a4f7281.xlsx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nMikihiro Fujiya and Hiroaki Konishi was funded by Kamui phama, Inc.","formattedTitle":"Splicing Factor 3a Subunit 1 Promotes Colorectal Cancer Growth via Anti-programmed cell death of Syntaxin12.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe treatment landscape for colorectal cancer (CRC) has significantly progressed through the development of molecularly targeted drugs, immune checkpoint inhibitors \u003csup\u003e1)2)\u003c/sup\u003e, and combination chemotherapy employing classical cytotoxic agents such as 5-FU. Despite these advancements, the mortality rate associated with advanced colorectal cancer remains elevated, underscoring the need for the identification and development of efficacious therapeutic targets\u003csup\u003e3)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecently, it has been reported that RNA-binding proteins (RBPs) influence cancer development through several mechanisms, including the stabilization of mRNAs and non-coding RNAs, as well as abnormal splicing in cancer cells. We have demonstrated that heterogeneous ribonucleoprotein (hnRNP) A1 exhibits oncogenic properties, which are inhibited by miR-18a, miR-26a, and miR-584 in colon cancer cells \u003csup\u003e4)5)\u003c/sup\u003e. Subsequently, hnRNP A1, hnRNP A2, and polypyrimidine tract-binding protein (PTB), highly induced in colorectal cancer cells \u003csup\u003e6)7)8)9)\u003c/sup\u003e, were reported to facilitate the alternative splicing of pyruvate kinase M1 (PKM1) to PKM2, thereby supporting tumor growth \u003csup\u003e10)\u003c/sup\u003e. Additionally, RBPs can promote tumorigenesis not only through abnormal expression but also via post-translational modifications such as phosphorylation \u003csup\u003e11)12)13)\u003c/sup\u003e. We observed that hnRNP A0 is specifically phosphorylated in cancer cells, and its phosphorylated form selectively binds to and stabilizes oncogenic RAB3GAP1 mRNA, resulting in the inhibition of tumor cell apoptosis and the promotion of colorectal cancer (CRC) development \u003csup\u003e6)\u003c/sup\u003e. Furthermore, through a high-throughput screening involving 1198 siRNAs targeting 416 human RBPs in colorectal, esophageal, and pancreatic cancer cells, coupled with comprehensive expression analysis using cDNA arrays, we identified 12 RBPs, including SF3A1, that undergo post-translational modifications in cancer cells, thereby promoting cancer cell growth \u003csup\u003e14)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSF3A1 is a subunit of the SF3a complex, an integral component of U2 snRNP, playing a crucial role in intron recognition and pre-RNA splicing during the splicing process\u003csup\u003e15)\u003c/sup\u003e. Notably, we observed that SF3A1 exerts its growth-promoting effects specifically in cancer cells, despite no discernible alteration in expression levels between tumor and normal cells \u003csup\u003e14)\u003c/sup\u003e. Genetic mutations in SF3A1 may play a role in pancreatic cancer development \u003csup\u003e16)\u003c/sup\u003e, and a reported correlation exists between SF3A1 gene polymorphisms and colorectal cancer (CRC) risk \u003csup\u003e17)\u003c/sup\u003e. However, the specific mRNAs with which SF3A1 interacts in CRC and whether this interaction is directly implicated in tumor growth remain unclear.\u003c/p\u003e \u003cp\u003eIn this study, our purpose was to elucidate the mechanisms of SF3A1-mediated tumor progression in colorectal cancer cells.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e・SF3A1 enhances the tumor progression in colorectal cancer cells, but not in non-cancerous cells\u003c/p\u003e \u003cp\u003eOur previous study suggested that 12 RBPs (RPS3, RBM22, EIF2S1, DHX8, RBM8A, UPF1, YBX1, SNRPE, SF3A1, U2AF1, SUPT6H, and EIF3G) exhibit tumor-promoting properties through posttranslational modifications in cancer cells\u003csup\u003e14)\u003c/sup\u003e. To validate the tumorigenic properties of each molecule in colorectal cancer cells, we conducted the sulforhodamine B (SRB) assay using the colorectal cancer cells, HCT116, and non-cancerous epithelial cells, HCEC-1CT, with downregulation of each RBP (Table\u0026nbsp;1). In this study, tumor-promoting RBPs were defined as RBPs that, when downregulated by siRNA, completely inhibit the proliferative potential of the cancer cell line HCT116 (resulting in \u0026gt;\u0026thinsp;100% growth inhibition) and have a slight effect (\u0026lt;\u0026thinsp;30% inhibition) on the proliferative potential of the non-tumor cell line HCEC-1CT. SF3A1 exhibited the highest promotion of tumor cell growth and the least effect on non-tumor cell growth. (Table\u0026nbsp;1). Downregulation of SF3A1 resulted in growth suppression in SW480 cells as well as HCT116 cells. No difference in SF3A1 mRNA expression was observed in colorectal cancer cells and non-cancerous epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, the knockdown efficacy was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). An analysis using GEPIA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.cn/\u003c/span\u003e\u003cspan address=\"http://gepia.cancer-pku.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) of 275 colorectal cancer tissue and 349 normal tissue showed that the expression of SF3A1 did not significantly change CRC patients (Supplemental Fig.\u0026nbsp;1). To explore the \u003cem\u003ein vivo\u003c/em\u003e tumor-promoting function of SF3A1, HCT116 cells were subcutaneously transplanted on the left and right sides of the backs of nude mice, and siRNA targeting SF3A1 or Scramble RNA was directly transfected into the transplanted HCT116 cells through local injection. The downregulation of SF3A1 significantly inhibited tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). These results suggest that SF3A1 exerts its growth-promoting effects selectively in cancer cells, despite no difference in SF3A1 expression levels between tumor and non-tumor cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e・SF3A1 promotes proliferation and inhibits programmed cell death (PCD) of colorectal cancer cells\u003c/p\u003e \u003cp\u003eTo elucidate the role of SF3A1 in cellular proliferation, immunocytochemistry for Ki-67 was performed in SF3A1-downregulated HCT-116 cells. The number of Ki-67-positive cells significantly decreased with the downregulation of SF3A1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Subsequently, to investigate whether SF3A1 inhibits programmed cell death, TUNEL staining was conducted. TUNEL-positive cells exhibited a significant increase in SF3A1-downregulated HCT116 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Meanwhile, the number of TUNEL and Ki-67 positive cells were not significantly changed in SF3A1-downregulated HCEC-1CT (Supplemental Fig.\u0026nbsp;2). Western blotting further revealed an elevation in PARP cleavage in SF3A1-downregulated HCT116 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These results strongly suggest that SF3A1 promotes the development of colorectal cancer cells by enhancing cell proliferation and inhibiting PCD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e・SF3A1 promotes the progression of colorectal cancer cells through stabilizing the mRNA of Syntaxin12 (STX12).\u003c/p\u003e \u003cp\u003eTo identify mRNAs directly interacting with SF3A1 in colorectal cancer cells, immunoprecipitation was conducted using an SF3A1 antibody to pull down SF3A1 along with its interacting RNAs from HCT116 cell lysates (Supplemental Fig.\u0026nbsp;3). Subsequently, transcriptome analysis was performed using RNA extracted from the precipitant after removing proteins and DNAs. A total of 7283 mRNAs were found to interact with SF3A1 in HCT116 cells (Supplemental Table\u0026nbsp;1). To identify mRNAs whose expressions were altered by the downregulation of SF3A1, transcriptome analysis was carried out in SF3A1-downregulated HCT116 cells, revealing that the expressions of 477 mRNAs significantly decreased upon SF3A1 downregulation (Supplemental Table\u0026nbsp;2). By integrating the IP-transcriptome and whole transcriptome analysis data, 144 mRNAs were identified as being stabilized through direct interacting with SF3A1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Among these, we selected the top 10 mRNAs exhibiting the most significant expression changes (Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine the association of these 10 mRNAs with tumor promotion in colorectal cancer cells, we generated siRNAs targeting each mRNA. Subsequently, each siRNA was transfected into HCT116 and HCEC-1CT cells, and their effects on tumor growth inhibition were analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The SRB assay revealed that downregulation of Syntaxin12 (STX12) exhibited cytotoxic effects in HCT116 but not in HCEC-1CT cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, the knockdown efficacy was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Additionally, RT-PCR demonstrated that STX12 mRNA was reduced by the downregulation of SF3A1 in HCT116, SW480, and HCEC-1CT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These results indicate that SF3A1 significantly contributes to the progression of colon cancer cells by stabilizing STX12 mRNA, whereas STX12 exhibits minimal impact on the growth of non-cancerous cells, specifically HCEC-1CT.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e・Syntaxin12 (STX12) inhibits the PCD in colorectal cancer cells\u003c/p\u003e \u003cp\u003eConsidering that SF3A1 promotes cell proliferation and inhibits PCD in colorectal cancer cells, we conducted immunocytochemistry for Ki-67 and TUNEL staining in STX12-downregulated cells. Contrary to expectations, immunocytochemistry for Ki-67 revealed that the number of proliferative cells remained unchanged upon the downregulation of STX12 in HCT116 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In contrast, TUNEL staining demonstrated a significant increase in DNA fragmented cells in the STX12-downregulated HCT116 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The number of TUNEL and Ki-67 positive cells were not changed in STX12 downregulated HCEC-1CT (Supplemental Fig.\u0026nbsp;4). Western blotting further indicated the induction of PARP cleavage in the STX12-downregulated HCT116 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These findings suggest that SF3A1 stabilizes STX12 mRNA, thereby exerting an anti-PCD, but not a proliferative, effect on colorectal cancer cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrated that the RNA-binding protein SF3A1 promotes tumor progression in both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models of colorectal cancer. Furthermore, we elucidated the tumor-promoting roles of SF3A1, mediating its direct interaction with STX12 mRNA, thereby facilitating its stabilization and suppressing PCD within colon cancer cells.\u003c/p\u003e \u003cp\u003eTUNEL staining revealed that SF3A1 possesses anti-PCD function against colorectal cancer cells. Our pull-down assay demonstrated that SF3A1 interacts to STX12 mRNA, and transcriptome analysis in SF3A1-downregulated cells revealed that the mRNA expression of STX12 was regulated by SF3A1. Furthermore, downregulation of STX12 was associated with an augmentation of PCD in colon cancer cells. These results clearly indicate that SF3A1 inhibits PCD in colon cancer cells by increasing the expression of STX12 mRNA.\u003c/p\u003e \u003cp\u003ePrevious investigations have indicated the tumor-promoting potential of STX12. Lee et al. demonstrated that STX12 is upregulated via the ROS/STAT3/NFE2L1 pathway, supporting tumor progression in hepatocellular carcinoma cells\u003csup\u003e18)\u003c/sup\u003e. STAT3 is a known key molecule involved in the development of colorectal cancer cells\u003csup\u003e19)20)\u003c/sup\u003e, and SF3A1 may promote colorectal cancer development by stabilizing STAT3-STX12-mediated antiapoptotic signaling. Additionally, STX12 suppresses the expression of miR-148a \u003csup\u003e21)\u003c/sup\u003e, which represses NFkB signaling, by binding to the 3'-UTR of TLR4 mRNA. Therefore, the STX12-miR-148a pathway also associates with tumor-promoting functions in colorectal cancer cells. Interestingly, bioinformatic analysis showed that the expression of STX12 was not significantly changed CRC patients (Supplemental Fig.\u0026nbsp;5). Malik et al. showed that STX12 is phosphorylated by SGK3\u003csup\u003e22)\u003c/sup\u003e, which is known as a downstream mediator of phosphatidylinositol 3-kinase (PI3K) oncogenic signaling\u003csup\u003e23)\u003c/sup\u003e, indicating that post-transcriptional modification, as well as the expression of STX12, plays a crucial role in colorectal cancer progression.\u003c/p\u003e \u003cp\u003eOur Ki67 staining revealed that SF3A1 possesses proliferative and anti-PCD functions against colorectal cancer cells, whereas STX12 exhibits only anti-PCD function, with less effect on cell proliferative capacity. In relation to the SF3A1-induced proliferation of colon cancer cells, our pull-down assay combined with transcriptome analysis identified various mRNAs, other than STX12, that interacted with SF3A1 and have cell proliferative properties, such as RASSF6 \u003csup\u003e24)\u003c/sup\u003e and SULF2 \u003csup\u003e25)\u003c/sup\u003e. This suggests that SF3A1 may promote cancer cell proliferation through stabilizing these mRNAs. Further analysis of the functions of these molecules, in which SF3A1 interacts to and stabilizes their mRNAs, is needed to identify the responsible molecules mediating the cell proliferation effect of SF3A1 in cancer cells.\u003c/p\u003e \u003cp\u003eThe SRB assay, TUNEL staining and Ki-67 staining revealed that the downregulation of SF3A1 significantly suppressed colorectal cancer cells while showing no impact on non-tumor cells. Notably, there was no significant expression change of SF3A1 between colorectal cancer and non-tumor cells. These observations suggest that SF3A1 undergoes specific post-translational modifications in cancer cells, thereby exerting oncogenic properties, including anti-PCD and proliferative functions. Phosphorylation is a well-known major type of post-translational modification that activates protein function. In our previous study, we demonstrated that hnRNP A0, an RNA-binding protein, stabilized oncogenic RAB3GAP1 mRNA via MAPKAP2 and supported tumor progression, specifically when Ser84 in hnRNP A0 was phosphorylated \u003csup\u003e13)\u003c/sup\u003e. SF3A1 possesses 23 serine/threonine residues, and the phosphorylation of these sites might be associated with SF3A1-induced stabilization of target mRNAs in colon cancer cells (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.phosphosite.org/proteinAction.action?id=4183\u0026amp;showAllSites=true\u003c/span\u003e\u003cspan address=\"https://www.phosphosite.org/proteinAction.action?id=4183\u0026amp;showAllSites=true\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Understanding the mechanisms of post-translational modification of SF3A1, including phosphorylation, ubiquitination, glycosylation, acetylation, and others, is expected to facilitate the development of therapeutic targets associated with SF3A1 protein. Several RNA-binding proteins (RBPs), including RBM22 \u003csup\u003e26)\u003c/sup\u003e and U2AF1 \u003csup\u003e27)\u003c/sup\u003e, along with SF3A1, have been reported to exert tumor-specific promotion, suggesting that these RBPs may also undergo post-translational modifications in cancer cells. However, the detailed target RNAs of these RBPs remain unknown. Our previous investigations have identified numerous RBPs upregulated in colorectal cancer cells, such as hnRNP A1, hnRNP H1, and RBMXL2. However, the main targets of mRNAs differ, and each RBP regulates distinct mechanisms, such as the cell cycle and sphingolipid metabolism in cancer cells\u003csup\u003e4)5)28)29)\u003c/sup\u003e. A detailed analysis of the mechanisms by which each RBP contributes to tumor-promoting effects is expected to facilitate the development of cancer therapy strategies based on changes in RBP expression and post-translational modifications.\u003c/p\u003e \u003cp\u003eDespite comparable knockdown efficiencies, the SRB assay also indicated that the inhibitory effect on cell proliferation observed in SW480 by SF3A1 suppression was less than in HCT116. This suggests the variation in the post-translational modification status of SF3A1 even among colon cancer cells. Studies aimed at elucidating the character of cancers that alter the status of post-translational modifications of SF3A1 will enable us to predict the efficacy of SF3A1-targeted cancer therapies.\u003c/p\u003e \u003cp\u003eIn this study, we have elucidated the functional alterations of SF3A1, an RNA-binding protein, in colorectal cancer cells, despite its comparable expression levels between tumor and non-tumor cells. This observation underscores its involvement in tumor growth. Furthermore, our findings reveal that SF3A1 plays a pivotal role in promoting tumor growth by exerting an anti-PCD effect through its direct interaction with STX12. Notably, inhibition of SF3A1 results in a significant suppression of tumor growth specifically in colorectal cancer cells, without affecting non-tumor cells. This study represents to identify the SF3A1-STX12 mRNA pathway as a potential therapeutic target for colorectal cancer.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eCell culture\u003c/p\u003e \u003cp\u003eHuman CRC cell lines (HCT116 and SW480) (American Type Culture Collection [ATCC]) were cultured in McCoy's 5A medium (HCT116) and Roswell Park Memorial Institute 1640 medium (SW480). The media were supplemented with 10% (vol/vol) fetal bovine serum (FBS) and 50 U/ml penicillin, and cells were maintained at 5% CO2 and 37\u0026deg;C under humidified conditions. Human colon epithelial cells (HCEC-1CT) (Summit Pharmaceuticals International Corporation) were cultured in ColoUp medium (DMEM/Medium 199 Earle\u0026rsquo;s, 4\u0026thinsp;+\u0026thinsp;1; Biochrom Cat# F0435 and Cat# FG0615). The medium included 4 mM GlutaMAXTM-1 (100\u0026times;, Gibco, Cat# 35050-038), 2% cosmic calf serum (Hyclone, Cat# SH30087), 20 ng/ml EGF (Sigma Aldrich, Cat# E9644), 10 \u0026micro;g/ml Insulin (Sigma Aldrich, Cat# I9278), 2 \u0026micro;g/ml Apo-Transferrin (Sigma Aldrich, Cat# T2036), 5 nM sodium-selenite (Sigma Aldrich, Cat# S5261), and 1 \u0026micro;g/ml hydrocortisone (Sigma Aldrich, Cat# H0396). The cells were maintained at 5% CO2 and 37\u0026deg;C under humidified conditions.\u003c/p\u003e \u003cp\u003esiRNA and Transfection\u003c/p\u003e \u003cp\u003e12 siRNAs targeting RBPs\u003csup\u003e14)\u003c/sup\u003e and a total of 18 siRNAs targeting 10 mRNAs, except for EIF4EBP3 (Table\u0026nbsp;2), were selected from the Silencer Select siRNA Libraries (Thermo Fisher Scientific) (Supplemental table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). EIF4EBP3 siRNA was generated by annealing two synthetic RNAs as follows: sense, 5'-UGGAUUAGAUGUCCAUUUCAA-3' and antisense, 5'-GAAAUGGACAUCUAAUCCAGU-3' (Hokkaido System Science Co., Ltd.). A negative control, consisting of a scrambled RNA sequence, was prepared by annealing two synthetic RNAs as follows: sense, 5'-UACGUACUAUCGCGCGGAU-3' and antisense, 5'-AUCCGCGCGAUAGUACGUA-3' (Hokkaido System Science Co., Ltd.). Transfection was carried out in triplicate using Lipofectamine RNAiMAX (Thermo Fisher Scientific) following the manufacturer's instructions for reverse transfection methods.\u003c/p\u003e \u003cp\u003eSRB assays\u003c/p\u003e \u003cp\u003eThe cells were seeded onto 96-well microplates at a density of 0.75 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well. Subsequently, the cells were fixed using a solution of 5% trichloroacetic acid (TCA) at 4 ℃ for one hour. Following fixation, the plates were rinsed four times with distilled water, dehydrated at room temperature, and then stained with 100 \u0026micro;L/well of 0.057% (wt/vol) sulforhodamine B (SRB) powder in 0.1% acetic acid. After staining, the microplates were washed four times with 0.1% acetic acid and re-dehydrated at room temperature. The cells were lysed in a 10 mM Tris-buffered environment, and the optical density (OD) was measured at a wavelength of 510 nm.\u003c/p\u003e \u003cp\u003eReal-time PCR\u003c/p\u003e \u003cp\u003eTotal RNA was isolated utilizing the RNeasy Mini Kit (Qiagen) following the manufacturer's instructions. cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). The Ct values were monitored across a spectrum using the Applied Biosystems 7300 Real-Time PCR system, employing Taqman gene expression assays for SF3A1 (Hs01066327) and STX12 (Hs00295291) in duplicate. The mRNA expression of 18S rRNA served as the normalization reference for each sample.\u003c/p\u003e \u003cp\u003eXenografts\u003c/p\u003e \u003cp\u003eThe dorsal lateral region of male BALB/c nude mice, aged 6 to 8 weeks, was subjected to a subcutaneous injection of HCT116 cells (2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells). After the tumor tissue reached 4\u0026ndash;5 mm, SF3A1 siRNA or scramble siRNA was administered daily using the GENOMONE-Si transfection kit (Ishihara Sangyo, Co, Ltd.) through local injection into the transplanted tumor. Tumor diameter was measured daily before transfection. The tumor sizes were calculated from digital caliper raw data using the following formula: Volume = (major tumor diameter) \u0026times; (minor tumor diameter).\u003c/p\u003e \u003cp\u003eImmunocytochemistry\u003c/p\u003e \u003cp\u003eHCT116 cells were cultured on glass chamber slides and subsequently fixed using a 4% paraformaldehyde solution at 4\u0026deg;C. The cells underwent washing with PBS, followed by permeabilization using a 0.1% Triton X-100 solution and subsequent blocking with 3% BSA in PBS. Subsequently, the slides were incubated overnight at 4\u0026deg;C with primary antibodies (Ki-67 [Novus, NB500-170]), followed by PBS washing and subsequent incubation with Alexa 488-conjugated secondary antibodies (ThermoFisher Scientific) for 1 hour at room temperature. The nuclei were counterstained with 4\u0026rsquo;,6-Diamidine-2\u0026rsquo;-phenylindole dihydrochloride (Sigma Aldrich).\u003c/p\u003e \u003cp\u003eTUNEL staining\u003c/p\u003e \u003cp\u003eHCT116 cells were cultured on glass chamber slides and subsequently fixed with a 4% paraformaldehyde solution at 4\u0026deg;C. The cells underwent PBS washing, followed by staining using an In Situ Cell Death Detection Kit with TMR red (Roche Diagnostic) according to the manufacturer's instructions. The cells were mounted with an anti-fade mounting medium, and immunofluorescence was visualized using a fluorescence microscope (KEYENCE Corporation).\u003c/p\u003e \u003cp\u003eRNA-immunoprecipitation\u003c/p\u003e \u003cp\u003eHCT116 cells were solubilized in NP-40 cell lysis buffer (Thermo Fisher Scientific), supplemented with a complete protease inhibitor cocktail (Merck) and RNasin (Promega Corporation). The lysates were centrifuged at 20,000\u0026times;g for 5 minutes to remove cellular debris. RNAs forming a complex with SF3A1 were selectively pulled down using either SF3A1 antibody (ThermoFisher Scientific ; A301-602A) or an isotype control antibody, employing a Dynabeads immunoprecipitation kit (VERITAS Corporation). The precipitated RNAs were isolated using phenol\u0026ndash;chloroform extraction and purified with a mirVana\u0026trade; Isolation Kit (ThermoFisher Scientific).\u003c/p\u003e \u003cp\u003eTranscriptome analyses\u003c/p\u003e \u003cp\u003eRNA libraries were generated using the Ion Total RNA-Seq Kit v2 (ThermoFisher Scientific) following the manufacturer\u0026rsquo;s instructions. The RNA libraries underwent an emulsion polymerase chain reaction (PCR) process utilizing the Ion OneTouchTM system and the Ion OneTouch 200 Template kit v3 (ThermoFisher Scientific). Template-positive Ion SphereTM particles were enriched and purified, prepared for subsequent sequencing using the Ion OneTouchTM ES system (ThermoFisher Scientific). Following this, the template-positive Ion SphereTM Particles were loaded onto Ion PI\u0026trade; Chips (ThermoFisher Scientific), and high-throughput sequencing was performed using the Ion Proton\u0026trade; Semiconductor sequencer (ThermoFisher Scientific). The entire sequencing dataset was aligned to the human reference genome sequence (GRCh37/hg19) using the Torrent Suite software program (ThermoFisher Scientific). After importing the expression data for each sample into the CLC Genomics Workbench software program (CLC bio, Aarhus, Denmark), distinctions among the samples were assessed using an unpaired t-test to determine their statistical significance.\u003c/p\u003e \u003cp\u003eWestern blotting\u003c/p\u003e \u003cp\u003eHCT116 cells were lysed in NP-40 cell lysis buffer (ThermoFisher Scientific) supplemented with cOmplete\u0026trade; Protease Inhibitor Cocktail (Merck). Following centrifugation at 20,000g for 5 minutes, the lysate was denatured with Laemmli Sample Buffer containing 2-mercaptoethanol at 95℃ for 5 minutes. Equal amounts of protein were loaded onto an SDS\u0026ndash;PAGE gel (12.5%), followed by transfer onto a nitrocellulose membrane at 100 V for 60 minutes. The blots were blocked in SuperBlock T-20 (PBS; ThermoFisher Scientific) for 1 hour, then incubated with the primary antibody in SuperBlock T‐20. The primary antibody used, specifically the cleaved poly-ADP-ribose polymerase (PARP) (#9546, Cell Signaling Technology, Inc.), was diluted to 1:1000 in SuperBlock T‐20 (PBS). Subsequently, an overnight incubation with the blots was conducted at 4℃. The blots were washed in 0.05% Tween20‐PBS (T‐PBS) three times for 15 minutes and then incubated in SuperBlock T‐20 (PBS) containing HRP-conjugated secondary antibodies (R\u0026amp;D Systems, Inc.). Following three washes in T-PBS for 15 minutes each, the blots were visualized using the Super Signal West Pico enhanced chemiluminescence system (ThermoFisher Scientific). Actin (612656, BD Transduction Laboratories) protein expression was used for normalizing protein levels.\u003c/p\u003e \u003cp\u003eStatistical Analyses\u003c/p\u003e \u003cp\u003eStatistical significance was assessed using Student's t-test, with p-values below 0.05 considered indicative of statistical significance.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eEthics Statement\u003c/h2\u003e \u003cp\u003eAnimal Studies: The studies were approved by the use of an opt-out methodology from the Medical Ethics Committee of Asahikawa Medical University (Approval No. R5-059).\u003c/p\u003e \u003c/div\u003e\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eMikihiro Fujiya and Hiroaki Konishi was funded by Kamui phama, Inc.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding Information\u003c/h2\u003e \u003cp\u003eThis paper was supported by Grants-in-Aid for Scientific Research, No. 21K07929 (M. Fujiya), 22K15363 (H. Konishi), 21KK0291 (H. Konishi) and 22K08047 (K. Moriichi), Intractable Disease Health and Labour Sciences Research Grants from the Ministry of Health, Labour and Welfare (M. Fujiya).\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eT.S. and H.K., and M.F. provided major input regarding the conceptual development of the studies, wrote the manuscript, and supervised all of the investigations. T.S., H.K, and H.T. performed the biochemical experiments. A.S., K.Y., K.T., K.A., N.U., S.K., K.M., H.T., and T.O. helped to design the studies, interpret the data, and prepare/review the manuscript. All the authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments:\u003c/h2\u003e \u003cp\u003eThe authors thank Chikage Yamamura, and Nobue Tamamura for their valuable technical assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMoreno, E.C., Pascual, A., Prieto-Cuadra, D., et al.: Novel Molecular Characterization of Colorectal Primary Tumors Based on miRNAs. Cancers (Basel). \u003cb\u003e11\u003c/b\u003e(3), 346 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/cancers11030346\u003c/span\u003e\u003cspan address=\"10.3390/cancers11030346\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLe, D.T., Uram, J.N., Wang, H., et al.: PD-1 Blockade in Tumors with Mismatch-Repair Deficiency. N Engl. J. 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Genes Dev. \u003cb\u003e33\u003c/b\u003e(9\u0026ndash;10), 482\u0026ndash;497 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1101/gad.319590.118\u003c/span\u003e\u003cspan address=\"10.1101/gad.319590.118\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e Epub 2019 Mar 6\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakahashi, K., Fujiya, M., Konishi, H., et al.: Heterogenous Nuclear Ribonucleoprotein H1 Promotes Colorectal Cancer Progression through the Stabilization of mRNA of Sphingosine-1-Phosphate Lyase 1. Int. J. Mol. 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PMID: 35384384; PMCID: PMC9554453\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable.1 SF3A1 has the least effect on non-tumor cells but the greatest effect on colon cancer cells.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"421\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003eHCEC-ICT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003eHCT116\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003eRPS3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e56%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e93%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003eRBM22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e35%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e110%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003eEIF2S1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e60%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e91%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003eDHX8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e21%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e88%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003eRBM8A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e71%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e120%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003eUPF1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e46%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e71%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003eYBX1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e39%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e66%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003eSNRPE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e45%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e112%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003eSF3A1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e29%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e111%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003eU2AF1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e24%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e80%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003eSUPT6H\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e50%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e105%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003eEIF3G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e40%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e97%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe inhibition rate of the SRB assay using the colorectal cancer cells, HCT116, and non-cancerous epithelial cells, HCEC-1CT, with downregulation of each RBP. The inhibition rate (%) = [1 \u0026ndash; (OD510 nm at day 3 of siRNA of each RBP- OD510 nm at day 1 of siRNA of each RBP) / (OD510 nm at day 3 of Scrambled RNA- OD510 nm at day 1 of Scrambled RNA)] \u0026times; 100. The cut-off for HCEC-1CT was defined as \u0026lt;30% and for HCT116 as \u0026gt;100%.\u003c/p\u003e\n\u003cp\u003eTable.2 The top 10 mRNAs with the largest changes.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"464\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.275862068965516%\" style=\"width: 27.6715%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"38.36206896551724%\" colspan=\"2\" rowspan=\"2\" style=\"width: 44.3113%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIP-Teranscriptome Analysis\u003cbr\u003e\u0026nbsp;(SF3A1/IgG)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.36206896551724%\" colspan=\"2\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eTranscriptome Analysis\u003cbr\u003e\u0026nbsp;(SF3A1/scramble)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" style=\"width: 27.6715%;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.225806451612904%\" style=\"width: 27.6715%;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.989247311827956%\" style=\"width: 21.7403%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFold Change\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.29032258064516%\" style=\"width: 24.1649%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ep-Value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.989247311827956%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFold Change\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.50537634408602%\"\u003e\n \u003cp\u003e\u003cstrong\u003ep-Value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.225806451612904%\" style=\"width: 27.6715%;\"\u003e\n \u003cp\u003eMGC32805\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.989247311827956%\" style=\"width: 21.7403%;\"\u003e\n \u003cp\u003e30.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.29032258064516%\" style=\"width: 24.1649%;\"\u003e\n \u003cp\u003e0.0008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.989247311827956%\"\u003e\n \u003cp\u003e-9.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.50537634408602%\"\u003e\n \u003cp\u003e0.013\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.225806451612904%\" style=\"width: 27.6715%;\"\u003e\n \u003cp\u003eDRICH1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.989247311827956%\" style=\"width: 21.7403%;\"\u003e\n \u003cp\u003e------\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.29032258064516%\" style=\"width: 24.1649%;\"\u003e\n \u003cp\u003e0.0246\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.989247311827956%\"\u003e\n \u003cp\u003e-8.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.50537634408602%\"\u003e\n \u003cp\u003e0.011\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.225806451612904%\" style=\"width: 27.6715%;\"\u003e\n \u003cp\u003eDNAJC5G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.989247311827956%\" style=\"width: 21.7403%;\"\u003e\n \u003cp\u003e7.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.29032258064516%\" style=\"width: 24.1649%;\"\u003e\n \u003cp\u003e0.0036\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.989247311827956%\"\u003e\n \u003cp\u003e-6.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.50537634408602%\"\u003e\n \u003cp\u003e0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.225806451612904%\" style=\"width: 27.6715%;\"\u003e\n \u003cp\u003eSLCO1B3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.989247311827956%\" style=\"width: 21.7403%;\"\u003e\n \u003cp\u003e61.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.29032258064516%\" style=\"width: 24.1649%;\"\u003e\n \u003cp\u003e0.0137\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.989247311827956%\"\u003e\n \u003cp\u003e-5.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.50537634408602%\"\u003e\n \u003cp\u003e0.012\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.225806451612904%\" style=\"width: 27.6715%;\"\u003e\n \u003cp\u003eGEMIN2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.989247311827956%\" style=\"width: 21.7403%;\"\u003e\n \u003cp\u003e3.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.29032258064516%\" style=\"width: 24.1649%;\"\u003e\n \u003cp\u003e0.0157\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.989247311827956%\"\u003e\n \u003cp\u003e-5.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.50537634408602%\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.225806451612904%\" style=\"width: 27.6715%;\"\u003e\n \u003cp\u003eEIF4EBP3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.989247311827956%\" style=\"width: 21.7403%;\"\u003e\n \u003cp\u003e6.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.29032258064516%\" style=\"width: 24.1649%;\"\u003e\n \u003cp\u003e0.0228\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.989247311827956%\"\u003e\n \u003cp\u003e-5.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.50537634408602%\"\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.225806451612904%\" style=\"width: 27.6715%;\"\u003e\n \u003cp\u003eMRPS31P\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.989247311827956%\" style=\"width: 21.7403%;\"\u003e\n \u003cp\u003e21.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.29032258064516%\" style=\"width: 24.1649%;\"\u003e\n \u003cp\u003e0.0090\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.989247311827956%\"\u003e\n \u003cp\u003e-5.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.50537634408602%\"\u003e\n \u003cp\u003e0.006\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.225806451612904%\" style=\"width: 27.6715%;\"\u003e\n \u003cp\u003eSTX12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.989247311827956%\" style=\"width: 21.7403%;\"\u003e\n \u003cp\u003e4.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.29032258064516%\" style=\"width: 24.1649%;\"\u003e\n \u003cp\u003e0.0069\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.989247311827956%\"\u003e\n \u003cp\u003e-4.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.50537634408602%\"\u003e\n \u003cp\u003e0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.225806451612904%\" style=\"width: 27.6715%;\"\u003e\n \u003cp\u003eVIM-AS1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.989247311827956%\" style=\"width: 21.7403%;\"\u003e\n \u003cp\u003e7.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.29032258064516%\" style=\"width: 24.1649%;\"\u003e\n \u003cp\u003e0.0139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.989247311827956%\"\u003e\n \u003cp\u003e-4.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.50537634408602%\"\u003e\n \u003cp\u003e0.023\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.225806451612904%\" style=\"width: 27.6715%;\"\u003e\n \u003cp\u003eLUCAT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.989247311827956%\" style=\"width: 21.7403%;\"\u003e\n \u003cp\u003e24.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.29032258064516%\" style=\"width: 24.1649%;\"\u003e\n \u003cp\u003e0.0011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.989247311827956%\"\u003e\n \u003cp\u003e-4.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.50537634408602%\"\u003e\n \u003cp\u003e0.012\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"RNA binding protein, splicing factor 3A1, syntaxin 12, colorectal cancer, programed cell death","lastPublishedDoi":"10.21203/rs.3.rs-4714660/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4714660/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRNA dysregulation mediated by abnormal RNA binding proteins (RBPs) is associated with tumorigenesis. However, the specific tumorigenic mechanisms of each RBP remained unclear. In this study, we demonstrate that splicing factor 3A1 (SF3A1) interacts to and stabilizes the mRNA of STX12, thereby inhibiting programmed cell death (PCD) in colorectal cancer cells. Downregulation of SF3A1 significantly inhibited cell growth in colorectal cancer cells, with minimal cytotoxicity observed in non-cancerous epithelial cells. We validated the tumor-promoting function of SF3A1 in an HCT116 transplanted mouse model. TUNEL staining and western blotting of PARP revealed SF3A1 inhibits PCD in colorectal cancer cells. A transcriptome analysis, combined with RNA-immunoprecipitation (IP), demonstrated that SF3A1 interact to and stabilized 144 mRNAs. Among these mRNAs, knockdown of STX12 (Syntaxin 12) in colorectal cancer cells inhibited cell growth but had no inhibitory effect on non-cancerous epithelial cells, HCEC-1CT. The mRNA levels of STX12 were significantly reduced upon downregulation of SF3A1, contributing to the inhibition of PCD in colorectal cancer cells. Therefore, SF3A1, which mediates STX12 mRNA stabilization, represents a promising therapeutic target for the treatment of colorectal cancer with fewer side effects.\u003c/p\u003e","manuscriptTitle":"Splicing Factor 3a Subunit 1 Promotes Colorectal Cancer Growth via Anti-programmed cell death of Syntaxin12.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-16 17:54:46","doi":"10.21203/rs.3.rs-4714660/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"76720142-b1d3-4ad4-8b42-b877fd4ddd23","owner":[],"postedDate":"September 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":36234370,"name":"Biological sciences/Cell biology/Cell death/Apoptosis"},{"id":36234371,"name":"Health sciences/Oncology/Cancer/Gastrointestinal cancer"}],"tags":[],"updatedAt":"2024-11-21T06:38:11+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-16 17:54:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4714660","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4714660","identity":"rs-4714660","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}