The Study On The Effects And Mechanisms Of Splicing Factor BUD31 Promoting Serous Ovarian Cancer Progression

Ovarian cancer is a common gynecologic malignancy and one of the three major malignancies of the female reproductive system.According to the statistical data of the Chinese population,there are about 52,100 new cases of ovarian cancer and 22,500 deaths per year,which are among the top 10 female tumors in terms of incidence and mortality.The mortality rate of ovarian cancer increases year by year,and the five-year survival rate of most patients is only 30%.According to histological typing,high-grade serous ovarian carcinoma(HGSOC)is the most important one.It is highly malignant,invasive and metastatic,and prone to chemotherapy resistance and recurrence.Due to the lack of early symptoms and early diagnosis,most patients are diagnosed at advanced stages at the first visit.Therefore,it is still an urgent task to study the mechanism of ovarian cancer development and explore novel and precise therapeutic tools.Splicing removes introns from pre-mRNA and ligating exons to form mature mRNA.The number of splicing events in tumors is more than 30%higher than in normal tissues.Splicing reprogramming can promote malignant behaviors such as malignant transformation,tumor proliferation,invasive metastasis,and drug resistance.The transregulatory element splicing factor stably regulates alternative splicing.Deregulated expression of splicing factor will lead to abnormal alternative splicing network.In addition,transcription and RNA splicing are mutually coupled to ensure orderly gene expression and regulation.Studies have shown that the knockdown of splicing factor BUD31 in MYC-activated mammary epithelial cells will lead to synthetic cell death.However,the biological function and clinical significance of BUD31 in tumors are unclear.Its regulation of alternative splicing events and downstream molecular networks has not been elucidated.This topic focuses on the role and molecular mechanism of BUD31 in ovarian cancer.It mainly includes the following three parts.Chapter 1 Expression,clinical significance and biological function of splicing factor BUD31 in serous ovarian cancerObjective:To screen for dysregulated splicing factors with prognostic relevance in ovarian cancer and investigate their functions.Methods:We analyzed differentially expressed genes in ovarian cancer based on TCGA and GTEx databases and validated their expression by qPCR and tissue microarray.We used the Kaplan-Meier Plotter and Qilu Hospital cohorts to perform prognosis and clinicopathological features analyses.The effects on the malignant behavior of ovarian cancer were explored using EdU,Transwell,and animal live imaging assays.Results:The results revealed that 37 out of 134 core splicing factors were aberrantly expressed in ovarian cancer,10 of which were significantly associated with progression-free survival and overall surviva1.BUD31,as one of them,was highly expressed and associated with a poor prognosis.Knockdown of BUD31 in HEYA8 and OV90 cells increased apoptotic cells,while overexpression inhibited hydrogen peroxideinduced apoptosis in ovarian cancer cells.EdU assay and Transwell assay demonstrated that overexpression of BUD31 in A2780 and OVCAR3 cells elevated the proliferation and invasive ability of ovarian cancer cells.In contrast,BUD31 knockdown in HEYA8 and OV90 cells had the opposite effect.Besides,overexpression of BUD31 in ID8 cells resulted in a significant increase in xenograft tumor mass and volume in vivo.Tumor load was lower in nude mice inoculated with BUD31-knockdown HEYA8 cells.Conclusion:Splicing factor BUD31 is highly expressed in ovarian cancer and is strongly associated with poor prognosis,exerts anti-apoptotic effects,and promotes proliferation and migration of ovarian cancer cells.Chapter 2 Mechanism of splicing factor BUD31 promoting ovarian carcinogenesis through regulation of BCL2L12 alternative splicingObjective:To investigate the mechanism by which BUD31 promotes malignant behavior in ovarian cancer and to elucidate the binding motifs of BUD31 on RNA and its splicing target genes.Methods:We analyzed BUD31-interacting proteins using Co-IP-MS.Further,RNA-seq was performed on HEYA8 cell lines with knockdown BUD31,and the length of the coding region,alternative splicing events,and regulated signaling pathways were analyzed.We performed SpyCLIP experiments to explore the binding motifs of BUD31.They were combined to analyze BUD31 directly regulated target genes.We verified the protein-RNA interaction using RIP-qPCR,RNA EMSA,and RNA pulldown.The halflife of transcripts was determined by RNA stability assay.Results:BUD31 interacts with U2 snRNP and HNRNP family proteins and participates in splicing regulation.BUD31 preferentially binds exon and intron regions near the splice site.Knockdown of BUD31 leads to extensive exon skipping and reduced abundance of transcripts in the long coding region and produces nonsense-mediated mRNA degradation(NMD)sensitive transcripts,leading to down-regulation of gene expression.Among 1408 genes with alternative splicing events and directly bound by BUD31,the anti-apoptotic gene BCL2L12 was screened out.BUD31 promoted exon 3 retention,reduced the proportion of NMD-sensitive transcripts,and up-regulated their expression by directly interacting with the precursor mRNA of BCL2L12.In vivo and in vitro experiments,overexpression of BCL2L12 partially reversed the inhibition of proliferation and clonogenic capacity of HEYA8 cells by knockdown of BUD31.Conversely,BCL2L12 knockdown impaired the anti-apoptotic effect of BUD31 on ovarian cancer.Conclusion:In summary,the splicing factor BUD31 exerts its oncogenic effects in ovarian cancer by directly regulating alternative splicing events such as exon skipping,causing altered expression of downstream target genes such as BCL2L12.Chapter 3 Mechanistic study of antisense oligonucleotides targeting BCL2L12 to inhibit malignant behavior in ovarian cancerObjective:To design antisense oligonucleotides targeting tumor-specific alternative splicing of BCL2L12 and verify its interference with exon skipping and its inhibitory effect on ovarian cancer.Methods:We designed antisense oligonucleotide(ASO)with phosphorothioate bond modification based on the binding signal of BUD31 near the BCL2L12 alternative exon.We used semi-quantitative PCR to verify the interfering effect of ASO on exon skipping and calculate EC50.qPCR and western blot were used to detect gene expression.MTT assay,EdU,flow cytometry,and TUNEL staining were used to verify the effect of ASO on ovarian cancer cells.Results:The expression of the anti-apoptotic gene BCL2L12 and the ratio of long and short transcripts were elevated in tumors and correlated with poor prognosis in TCGA and GTEx databases.ASO2 was designed to target BCL2L12 alternative splicing.Exon 3 skipped and produced a short transcript of BCL2L12 after treatment of ovarian cancer cells A2780 and HEYA8 with ASO2.The short transcript was then degraded through the NMD pathway and caused down-regulation of BCL2L12 expression.Increased apoptotic cells and up-regulation of Cleaved-Caspase3 protein expression were detected.Conclusion:Antisense oligonucleotides can mediate exon 3 skipping of BCL2L12,thus inducing apoptosis in ovarian cancer cells.In conclusion,this study is the first to report that BUD31 promotes the development of serous ovarian cancer.The splicing factor BUD31 is highly expressed in ovarian cancer and is associated with a poor prognosis.BUD31 promotes proliferation,migration,and tumorigenesis of ovarian cancer cells in vivo and enhances their antiapoptotic ability.Mechanistic studies have shown that BUD31 binds mainly to exons and introns near precursor mRNA splice sites,which regulates exon skipping and shortening coding region length.Furthermore,ASO targeting alternative splicing of BCL2L12 inhibits ovarian cancer proliferation and induces apoptosis.Our study provides new targets and strategies for diagnosing and treating ovarian cancer.Innovations of this study1.This study combined the molecular mechanism of ovarian cancer development with alternative splicing and confirmed the relationship between splicing factor BUD31 and poor patient prognosis.2.This study elucidated the downstream molecular network of BUD31-regulated splicing using combined SpyCLIP-seq and RNA-seq analysis and identified RNAbinding motifs on the whole genome.3.In this study,antisense oligonucleotides targeting BCL2L12 exon skipping were designed and confirmed to inhibit proliferation and induce apoptosis in ovarian cancer cells.Shortcomings of this study1.Cell lines used in this study did not possess the typical molecular features of highgrade serous ovarian cancer,and farther validation is needed in the future to explore the splicing factors and alternative splicing events specific to high-grade serous ovarian cancer.2.The control samples for ovarian cancer in the public database used in this study were mostly normal ovarian tissues,and sequencing data of normal ovarian epithelium after oviductal parachute or mierodissection need to be added in the future to be used for more accurate screening of differentially expressed genes in ovarian cancer3.This study failed to explore the effects of BUD31 on ferroptosis,pyroptosis,angiogenesis,cell autophagy,and homologous recombination repair in ovarian cancer cells.