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

doi:10.21203/rs.3.rs-4714660/v1

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

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 targets³⁾.

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 process¹⁵⁾. 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.

・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 cells¹⁴⁾. 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. 1A, 1B, the knockdown efficacy was shown in Fig. 1C). 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. 1D). 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. 2A). 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. 2B). 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. 2C). 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. 3A). 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. 3B). The SRB assay revealed that downregulation of Syntaxin12 (STX12) exhibited cytotoxic effects in HCT116 but not in HCEC-1CT cells (Fig. 4A, the knockdown efficacy was shown in Fig. 4B). Additionally, RT-PCR demonstrated that STX12 mRNA was reduced by the downregulation of SF3A1 in HCT116, SW480, and HCEC-1CT (Fig. 4C). 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. 5A). In contrast, TUNEL staining demonstrated a significant increase in DNA fragmented cells in the STX12-downregulated HCT116 cells (Fig. 5B). 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. 5C). These findings suggest that SF3A1 stabilizes STX12 mRNA, thereby exerting an anti-PCD, but not a proliferative, effect on colorectal cancer cells.

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 cells¹⁸⁾. STAT3 is a known key molecule involved in the development of colorectal cancer cells¹⁹⁾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 SGK3²²⁾, which is known as a downstream mediator of phosphatidylinositol 3-kinase (PI3K) oncogenic signaling²³⁾, 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.

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 RBPs¹⁴⁾ 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 × 10⁴ 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 × 10⁶ 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.

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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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

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 <30% and for HCT116 as >100%.

Table.2 The top 10 mRNAs with the largest changes.

	IP-Teranscriptome Analysis (SF3A1/IgG)		Transcriptome Analysis (SF3A1/scramble)
	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

Yes there is potential Competing Interest. Mikihiro Fujiya and Hiroaki Konishi was funded by Kamui phama, Inc.

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

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