The Study On The Mechanism Of Splicing Factor SF3B4 Promoting The Development Of Serous Ovarian Cancer

Ovarian cancer(OC)is the most lethal gynecologic malignancy,although its morbidity is lower than cervical and endometrial cancer.More than 60%of the patients were diagnosed at stage Ⅲ and stage Ⅳ due to morbidity concealment,lack of early diagnosis technology and rapid progress.Because of lack of lasting and effective therapeutics,the five-year survival rate was about 45%.Recent years,although the advent of antiangiogenic drugs and PARP inhibitors can benefit some patients,but they can’t change the treatment outcome of all ovarian cancer patients fundamentally.Therefore,the mechanism of initiation and development of ovarian cancer is necessary to be elucidated further.Number of proteins in human is far more than the number of coding genes due to alternative splicing.Abnormal splicing is according to many human diseases.In malignant tumors,the level of alternative splicing increased significantly.RNA binding proteins(RBPs)play an important role in alternative splicing.Studies have shown that RBPs can be involved in proliferation,invasion,apoptosis,angiogenesis and drug resistance.RBPs are key factors in regulating tumor development.SF3B is important for pre-mRNA splicing.Previous research have shown that SF3B1,SF3B2,SF3B3 can regulate target genes’ expression by alternative splicing to promote cancer progression.Importantly,SF3B1 inhibitor FR901464 plays an anticancer effect in variety of cancers.Through the analysis of TCGA database,we found that SF3B4 was significantly increased and related to worse prognosis of ovarian cancer patients.SF3B4 was confirmed to be not only as a component for pre-mRNA splicing,but also as a regulator for transcription,translation,and cell signaling.SF3B4 promotes cell proliferation and metastasis of hepatocellular carcinoma through regulating KLF4.In esophageal squamous cell carcinoma,SF3B4 plays an oncogenic role.On the contrary,SF3B4 acts as cancer suppressor gene in pancreatic cancer.However,the underlying mechanism of SF3B4 in the progression of ovarian cancer remained to be elucidated.In this study,we elaborate the expression,clinical significance and biological function of SF3B4 in ovarian cancer.The upstream and downstream regulation mechanism was also explored.The research content is divided into the following three parts:1,Study on biological function and clinical significance of SF3B4 in serous ovarian cancer.2,Screening of downstream splicing events of SF3B4 and study on the biological function of downstream target.3,Study on the upstream mechanism of SF3B4 in ovarian cancer.Part Ⅰ:Study on biological function and clinical significance of SF3B4 in serous ovarian cancerPurposeThe expression of SF3B4 in ovarian cancer was detected to explore the relationship between its expression level and clinicopathological features and prognosis of ovarian cancer patients.To investigate the effects of SF3B4 on malignant biological behaviors of OC cells.MethodsTCGA and RBPDB database was used to analyze the abnormally high expression RNA-binding proteins in serous ovarian cancer.CSIOVDB database was used to analyze RNA-binding proteins which was related to prognosis of ovarian cancer patients.TCGA database was used to analyze the expression of SF3B4 in various cancer types.TCGA-GTEX database was used to analyze SF3B4 mRNA expression of ovary,fallopian tube(FT)and high grade serous ovarian cancer(HGSOC)samples.CPTAC-JHU database was used to analyze SF3B4 protein expression of HGSOC and ovary tissues.TCGA database was used to analyze the amplification of SF3B4 DNA in HGSOC and the relationship with SF3B4 mRNA.K-M Plotter and CSIOVDB was used to analyze the relationship between SF3B4 and prognosis in ovarian cancer patients.qRT-PCR and Western-Blot was used to detect SF3B4 mRNA and protein expression in serous ovarian cancer(SOC)and FT.The expression of SF3B4 in clinical samples was detected by immunohistochemistry and the relationship with clinicopathological features was further analyzed.Ovarian cancer cell lines with SF3B4 stable up-regulated and down-regulated were constructed by retrovirus infection.MTT,EdU,colony formation and Transwell assay were used to detect the effect of SF3B4 on the proliferative and invasion ability of OC cells in vitro.The effect of SF3B4 on tumorigenesis ability of OC cells in vivo was detected by tumor xenograft models.Results1.SF3B4 is overexpressed in ovarian cancer and correlates with poor prognosisThe analysis of TCGA and CSIOVDB database showed that SF3B4,IGF2BP2 and IGF2BP3 were up-regulated obviously and related to poor prognosis in ovarian cancer patients.Among them,only SF3B4 was core splicing factor.The analysis of TCGA-GTEX showed that the expression of SF3B4 mRNA is much higher in HGSOC than ovary and FT tissues.The analysis of CPTAC-JHU showed that the expression of SF3B4 protein is much higher in HGSOC than FT samples.The analysis of CSIOVDB database showed that increased expression of SF3B4 was correlated with poor prognosis.2.SF3B4 is overexpressed in ovarian cancer tissues and cell linesWe validated SF3B4 expression by qRT-PCR and Western Blot(WB)in fresh-frozen tissues,it was frequently up-regulated in serous ovarian cancer samples compared with FT tissues.The protein expression of SF3B4 in HEY,A2780 and SKOV3 cells was much higher than that in FT187 cells.3.SF3B4 is associated with CA125 level,omental metastasis and platinum resistance in ovarian cancer patientsIHC was performed on 117 SOC tissue samples and the result showed that SF3B4 expression was significantly correlated with age(p=0.018),serum CA125 level(p=0.009),ascites(p=0.029),omentum metastasis(p=0.012)and platinum sensitivity(p=0.049).4.OC cell lines with SF3B4 stable up-regulated and knockdown was constructedWe constructed SF3B4 stable overexpression cell lines in SKOV3 and stable low-expression cell lines in HEY qRT-PCR and WB was used to detect the efficiency of overexpression and the result showed that SF3B4 was overexpressed about 5-fold.qRT-PCR and WB was used to detect the efficiency of knockdown and the result showed that the level of SF3B4 mRNA decreased about 90%and SF3B4 protein decreased about 70%.5.SF3B4 promotes the proliferation and colony formation ability of OC cells in vitroThe results of MTT assay showed that the proliferation rate of OC cells was significantly decreased from the third day after SF3B4 knockdown.On the contrary,the proliferation rate of SKOV3 cells was significantly increased.A clonogenic assay was performed,and we found that SF3B4 dramatically promoted the clonogenic capacity of SKOV3 cells.In contrast,SF3B4 knockdown led to reduced clonogenicity.6.SF3B4 promotes the mobility ability of OC cells in vitroWe measured migration and invasion ability with a Transwell assay.The results showed that downregulation of SF3B4 decreased the migration and invasion ability of OC cells,whereas upregulation of SF3B4 had the opposite effect.7.SF3B4 promotes tumorigenic ability of OC cells in vivoIn vivo,we found that knockdown of SF3B4 reduced the growth of ovarian cancer cells significantly.The size(0.13 cm3 vs 0.36cm3)and weight(0.177g vs 0.385g)of xenograft tumors was much smaller and lighter in SF3B4-konckdown group than control group.Similarly,the level of Ki-67 was significantly decreased in SF3B4-konckdown group than control group.ConclusionSF3B4 is overexpressed in serous ovarian cancer and its high expression is associated with poor prognosis.SF3B4 promotes proliferation,clone formation,migration and invasion ability of ovarian cancer cells in vitro and in vivo.SF3B4 can be used as a prognostic biomarker and a potential therapeutic target for ovarian cancer patients.Part Ⅱ:Screening of downstream splicing events of SF3B4 and study on the biological function of downstream targetPurposeRNA-binding proteins regulate the expression of downstream target genes by alternative splicing.In this part,to explore the mechanism of SF3B4 promoting ovarian cancer malignancy,RNA-seq was used to screen downstream target genes and differentially splicing event of SF3B4 knockdown.Then,the biological function of its downstream target gene in ovarian cancer was explored.MethodsSmall interfering RNA(siRNA)was used to knockdown the expression of SF3B4 in ovarian cancer cells.RNA-seq was used to analyze the differentially expressed genes(DEGs)and alternative splicing(AS)events and screen downstream target gene.qRT-PCR was used to verify regulatory relationship of partial DEGs.AS events were analyzed by R langue and rMATS software.Ensembl website was used to enquiry and compare different transcripts of target gene.The specific primers of different transcripts of target gene were designed.RT-PCR was used to detect the splicing efficiency of target gene after SF3B4 knockdown.RIP-PCR was used to verify the direct combination of SF3B4 and target gene.qRT-PCR and western blot was used to detect mRNA and protein expression of target gene upon SF3B4 knockdown.Expression level of different transcripts was analyzed by TCGA-GTEX and the correlation with SF3B4 was aslo analyzed in ovarian cancer.K-M software was used to analyze the relationship between the main transcripts of target gene and the prognosis of ovarian cancer patients.siRNA was used to decrease the expression of downstream target gene.MTT,EdU,colony formation and transwell assay were used to detect the effect of target gene on the proliferative and invasion ability of OC cells.We knockdown of downstream target gene in SF3B4-overexpressed SKOV3 cell line to verify the effect of downstream gene on the cancer promoting capacity of SF3B4.Results1.Analysis and verification of RNA-seq resultsThe result of RNA-seq showed that there were 665 upregulated and 501 downregulated genes.GO analysis showed that the biological processes were enriched in regulation of transcription,extracellular matrix organization and cell adhesion.KEGG pathway analysis showed that the pathways were enriched in the TNF signaling pathway,cell cycle and DNA replication.We verified 5 DNA replication pathway-related genes(DNA2,MCM6,POLA1,RFC3,PRIM1)after SF3B4 knockdown in HEY cells through qRT-PCR,and the results showed that 5 selected genes were obviously downregulated,which was consistent with the RNA-seq results.2.Screening of downstream target genes of SF3B4 in OCThe result of RNA-seq showed that there were 2376 skipped exons and 320 retained introns after SF3B4 knockdown in HEY cells.940 genes were identified to be differential expressed in TCGA OC and involved in the differential alternative splicing events.Studies have shown that SF3B family could regulate the expression of downstream genes by inclusion of intron.So,we identified 27 critical genes both involved in intron retention events after SF3B4 knockdown(p<0.05,|delInclevel|>0.1)and positively correlated with SF3B4 expression in TCGA TARGET GTEx cohort(Pearson r≥0.4,p<0.05).The results of qRT-PCR showed that 6 genes(DDX11,FNBP1L,PITPNM1,DDX51,PKD1 and RAD52)of these 27 genes were obviously downregulated after SF3B4 knockdown in HEY.RAD52 is involved in DNA damage repair and its inhibitor can improve therapeutic effect of BRCA-deficient malignancies.Therefore,we selected RAD52 for further research.3.SF3B4 regulates alternative splicing of RAD52We used R langue and rMATS software to visualize the RNA-seq reads of DEGs in response to SF3B4 knockdown in HEY cells,and intron retention of RAD52 was found to be obviously increased.Retained intron 8 of protein-coding transcript variant RAD52-202 was verified by Ensembl genome browser.The result of RT-PCR showed that knockdown of SF3B4 significantly reduced the protein-coding transcript variant RAD52-202 and increased the noncoding transcript variant RAD52-206.The result of RIP-PCR showed that SF3B4 protein could bound to RAD52 mRNA specifically.Moreover,knockdown of SF3B4 obviously decreased the expression of RAD52 mRNA and protein in OC cells.4.RAD52 is overexpressed in OC and correlates with poor prognosisThe analysis of TCGA and GTEX databases showed that RAD52-202 transcript(spliced)was obviously upregulated and noncoding transcript variant RAD52-206(unspliced)was obviously downregulated in most OC samples.Kaplan-Meier analysis revealed that overexpression of RAD52-202 transcript indicated poor OS in OC patients.5.OC cell lines with RAD52 stable up-regulated and knockdown was constructedWe constructed RAD52 stable overexpression cell lines in HEY.qRT-PCR and WB was used to detect the efficiency of overexpression and the result showed that RAD52 was overexpressed about 300-fold.siRNA was used to decrease the expression of RAD52 in HEY,A2780 and SKOV3.qRT-PCR and WB was used to detect the efficiency of knockdown and the result showed that the level of RAD52 mRNA decreased about 70%and RAD52 protein decreased about 90%.6.RAD52 promotes the proliferation ability of OC cells in vitroThe results of MTT assay showed that the proliferation rate of OC cells was significantly decreased from the second day after RAD52 knockdown.The results of EdU assay showed that the percentage of dividing cells decreased about 15%-20%after RAD52 knockdown.A clonogenic assay was performed,and we found that knockdown of RAD52 dramatically weakened the clonogenic capacity of OC cells.7.RAD52 promotes the mobility ability of OC cells in vitroWe measured migration and invasion ability with Transwell assay.The result showed that number of crossed cells was decreased about 50%after RAD52 knockdown.8.Knockdown of RAD52 impaired the phenotype of SF3B4 overexpressionThe results of rescue assays showed that knockdown of RAD52 in SF3B4-overexpressing OC cells successfully impaired the enhanced capacity of cell proliferation,migration and invasion caused by SF3B4.ConclusionThe splicing efficiency of RAD52 mRNA is increased when SF3B4 expression is upregulated.RAD52 is overexpressed in serous ovarian cancer.RAD52 promotes proliferation and invasion ability of ovarian cancer cells in vitro.Knockdown of RAD52 impaired the enhanced ability of cell proliferation and invasion caused by SF3B4.SF3B4 promotes malignant behavior of ovarian cancer by regulating RAD52.Part Ⅲ:Study on the upstream mechanism of SF3B4 in serous ovarian cancer PurposeThe expression of gene is correlated with its amplification.Similarly,the transcription and post-transcriptional regulation of gene is also important to its expression.miRNAs are small noncoding RNAs,they regulate gene expression by binding to the 3’UTR of target genes.Transcription factors(TF)regulate gene transcription by binding to specific regulatory sequences.In this part,the upstream regulatory mechanism of SF3B4 overexpression was elucidated,and the effects of upstream regulatory molecule on biological behavior of ovarian cancer were clarified.MethodsmiRNAs that might bind to the 3’UTR of SF3B4 were predicted through StarBase.miRNAs that markedly downregulated were analyzed through GSE47841 database.Then,the miRNA might regulate the expression of SF3B4 were screened.The binding site of upstream miRNA in SF3B4 3’UTR was predicted by StarBase,and the wild type and mutant potential binding site was constructed.A dual luciferase reporter gene assay was used to confirm whether the upstream miRNA could bind to SF3B4 mRNA 3’UTR directly.qRT-PCR was used to verity the effect on miRNA and SF3B4 mRNA expression after transfecting miRNA mimics transiently.Western Blot was used to detect SF3B4 protein expression after transfecting miRNA mimics transiently.MTT,EdU,colony formation and transwell assay were used to detect the effect of miRNA mimics on the proliferative and invasion ability of OC cells.We transiently transfected miRNA mimics and SF3B4 plasmid into SKOV3 cells to confirm whether SF3B4 could reverse the effect of miRNA mimics.Transcription factors that might bind to the promoter of SF3B4 were predicted through AnimalTFDB 3.0.Genes that positively correlated with SF3B4 were analyzed through TCGA.Then,the transcription factor might regulate the expression of SF3B4 were screened.The promoter of SF3B4 was cloned into pGL 4.26 vector.A dual luciferase reporter gene assay was used to confirm whether the upstream transcription factor could activate the SF3B4 promoter.Upstream transcription factor stable up-regulated and knockdown OC cell lines was constructed.qRT-PCR and WB was used to detect the efficiency of overexpression and knockdown and the effect to SF3B4 mRNA and protein.The effect of transcription factor on the proliferative capaity of OC cells was detected by MTT,EdU and colony formation assay.The effect of TF on the invasion capacity of OC cells was detected by transwell assay.We knockdown of SF3B4 in transcription factor-overexpressed HEY cell line to verify the effect of SF3B4 on the cancer promoting capacity of upstream transcription factor.Results1.Screening of upstream posttranscriptional regulators of SF3B4 in OCWe found 34 miRNAs that might bind to the 3’UTR of SF3B4 through StarBase.Then,we found that 121 miRNAs were downregulated in OC tissues from GSE47841.Overlapping analysis found that only miR-509-3p matched the filter conditions.The result of dual luciferase reporter gene assay showed that increased miR-509-3p decreased the luciferase activity significantly in wild type 3’UTR.2.miR-509-3p negatively regulates the expression of SF3B4The result of qRT-PCR showed that the expression of miR-509-3p significantly upregulated after transiently transfecting miRNA mimics into OC cells.The results of qRT-PCR and WB showed that SF3B4 mRNA and protein expression were significantly decreased when we transiently transfected OC cells with miR-509-3p mimics.3.miR-509-3p inhibits the proliferation ability of OC cells in vitroMTT and EdU assay showed that the proliferation capacity decreased obviously after transiently transfecting miR-509-3p mimics in OC cells.The results of clone formation assay were consistent with MTT and EdU.4.miR-509-3p inhibits the mobility ability of OC cells in vitroTranswell assay showed that the number of crossed cells was decreased obviously after transiently transfecting miR-509-3p mimics.5.SF3B4 rescued the inhibitory effect of miR-509-3p on the proliferation and mobility ability of OC cellsWe constructed four groups of SKOV3 cells with different expression levels of miR-509-3p and SF3B4 to perform rescue assay.The results of rescue assays showed that upregulation of SF3B4 reversed the inhibitory effect of miR-509-3p in SKOV3 cells.6.Screening of upstream transcriptional regulators of SF3B4 in OCWe found 509 transcription factors that might bind to the promoter of SF3B4 by AnimalTFDB 3.0.Then,we found 32 genes that positively correlated with SF3B4 by TCGA.Overlapping analysis found that only SETDB1 matched the filter conditions.The result of luciferase assay showed that the luciferase activity of SF3B4 promoter significantly decreased after SETDB1 knockdown.7.SETDB1 positively regulates the expression of SF3B4We constructed SETDB1 stable overexpression cell lines in HEY.qRT-PCR and WB was used to detect the efficiency of overexpression and the result showed that SETDB1 was overexpressed about 6-fold.SETDB1 and SF3B4 mRNA and protein expression were significantly decreased when we transiently transfected OC cells with SETDB1 siRNA.8.SETDB1 promotes the proliferation ability of OC cells in vitroThe results of MTT assay showed that the proliferation rate of OC cells was significantly decreased from the third day after SETDB1 knockdown.On the contrary,the proliferation rate of HEY cells was significantly increased from the third day after SETDB1 upregulated.The results of EdU assay showed that the percentage of dividing cells decreased about 10%-15%after SETDB1 knockdown.A clone formation assay was performed,and we found that the clone number of OC cells was decreased about 50%after SETDB1 knockdown.9.SETDB1 promotes the mobility ability of OC cells in vitroTranswell assay showed that the number of crossed cells was decreased about 50%after SETDB1 knockdown.10.Knockdown of SF3B4 impaired the phenotype of SETDB1 overexpressionThe results of rescue assays showed that knockdown of SF3B4 in SETDB1-overexpressing HEY cells successfully impaired the enhanced capacity of cell proliferation,migration and invasion caused by SETDB1.ConclusionThe expression of miR-509-3p is downregulated in ovarian cancer cells.miR-509-3p binds to SF3B4 3’UTR directly and regulates its expression negatively.miR-509-3p inhibits malignant behaviors of ovarian cancer cells and SF3B4 can reverse its inhibitory effect.The expression of SETDB1 is upregulated in ovarian cancer cells.SETDB1 binds to SF3B4 promoter and positively regulates the expression of SF3B4.SETDB1 promotes malignant behaviors of ovarian cancer cells and knockdown of SF3B4 impaired the phenotype of SETDB1 overexpression.Innovation of this study1,In this study,we explored the differentially expressed splicing factors in ovarian cancer and clarified the expression and clinical significance of SF3B4 in ovarian cancer.2,We confirmed the biological function of SF3B4 in ovarian cancer for the first time.3.Through RNA-seq analysis,RAD52 was found to be the direct downstream target gene of SF3B4 for the first time.SF3B4 mediated malignant behavior of ovarian cancer through regulating alternative splicing of RAD52.4,We clarified the upstream regulatory mechanism of SF3B4 and proved that miR-509-3p negatively regulated SF3B4 expression by directly binding to its mRNA.SETDB1 positively regulated SF3B4 expression by binding to its promoter.Limitations of this study1,The effect of SF3B4 on drug resistance of OC cells have not been studied.2,In this study,we discussed the effect of SF3B4 on the xenogenic subcutaneous tumorigenicity of ovarian cancer cells,but abdominal tumorigenesis and PDX animal models were not established to further explore the biological function of SF3B4 in vivo.