Splicing Factor PQBP1 Curtails BAX Exon Inclusion To Promote Ovarian Carcinogenesis And Its Targeted Strategy

Ovarian cancer(OV)is the most difficult malignant tumor to treat and seriously threatens the life and health of women.A lack of symptoms for early diagnosis,the complexity of types for accurate classification,and relapse and drug resistance are the three major problems recognized worldwide.The five-year overall survival rate is approximately 30%-40%,which has not changed for more than 100 years.RNA splicing is the process of removing precursor mRNA introns,retaining exons,and forming a 3’ terminal end through polyadenylation.Alternative splicing(AS)is an important component of RNA splicing,which can be divided into exon skipping(SE),intron rentention,mutually exclusive exons,alternative 5’ or 3’splicing site,etc.The number of splicing events in tumors is more than 30%higher than that in normal tissues.Abnormal splicing of precursor mRNA can promote the malignent behaviors of tumors.However,due to the complexity of splicing types,the diversity of alternative splicing genes and the limitations of research methods,abnormal splicing events in tumors and their cancer-promoting mechanisms have not been well clarified and are worthy of further exploration.This paper focuses on the role of the X-linked intellectual deficiency-related protein,polyglutamine-binding protein 1(PQBP1)in ovarian cancer;we adopted the integrated RNA omics method to comprehensively explore its role as a splicing factor to promote the development of tumors and constructed an alternative spliced gene network regulated by PQBP1.The application of antisense oligonucleotide(ASO)drugs targeting alternative splicing of downstream key genes in tumor therapy was also studied.This paper mainly consists of the following three parts:Chapter Ⅰ:Expression of PQBP1 in ovarian cancer and its clinical significance and effects on the biological behavior of cancer.By transcriptome sequencing and proteomics sequencing,we found that PQBP1 was highly expressed at both mRNA and protein levels in a variety of tumors.Approximately 10%of ovarian cancer patients show PQBP1 copy number amplification.Furthermore,expression of PQBP1 correlates positively with tumor mutation burden(TMB).Subsequently,expression was verified through public databases and the Qilu specimen database,and prognostic analysis and univariate and multivariate Cox regression analyses were performed,which suggested that high expression of PQBP1 was associated with the staging of ovarian cancer and greater omental metastasis.High PQBP1 expression and platinum resistance are independent risk factors for reduced overall survival in ovarian cancer.Based on the above studies,we constructed ovarian cancer cell lines with PQBP1 knockdown and overexpression and studied the effects of PQBP1 on the malignant biological behavior of the cells.After knocking down PQBP1 with DOX induction,the proliferation ability and the invasion and migration abilities of ovarian cancer cells decreased;in addition.subcutaneous and abdominal tumorigenesis ability decreased significantly in nude mice,the tumor growth rate slowed,and the tumor weight was significantly lower than that in the control group.Overexpression of PQBP1 had the opposite effect.Cell and animal experiments confirmed that PQBP1 can act as an oncogenic gene by promoting the proliferation of tumor cells and increasing tumorigenesis in vivo.Chapter Ⅱ:PQBP1 inhibited OV cell apoptosis by directly binding to BAX and regulating its exon 2 skipping.We further explored the binding preference of PQBP1 as a splicing factor to pre-mRNA and its regulation of splicing events.Using RNA-seq and SpyCLIP-seq technology,PQBP1 was found to mainly bind to the exon region and near the splicing site,which is the junction of exon and intron,and regulate exon skipping events.Full-length transcriptome sequencing and RIP-seq were adopted to verify these results.Exon and intron enrichment analysis showed that after PQBP1 knockdown,introns near the 3’ end of exon skipped and the number of exon skip events increased.Furthermore,PQBP1 regulated gene expression by direct binding to it,and the intensity of regulation was related to the abundance of binding peaks and the location of binding.KEGG pathway analysis showed that PQBP1 directly binding and regulating splicing genes were enriched in apoptosis,RNA degradation,RNA transport and mRNA surveillance pathways.Among the target genes directly regulated by PQBP1,we focused on apoptosis regulator,BAX(BCL2 Associated X),a proapoptotic gene of the BCL2 family,the exon 2 skipping of which produces the nonsense degraded short transcript BAX-S.Sequencing data showed that PQBP1 promotes skipping of BAX exon 2 by direct binding to the region of BAX exon 2 and intron 1,reducing the content of full-length BAX transcript(BAX-L)and increasing the content of BAX-S,thus reducing expression of BAX.Minigene,RIP-qPCR,RNA pull-down and RNA half-life assays further verified the conclusion.TCGA,GSEA database and qPCR analysis of specimens from Qilu Hospital showed that the short and long transcripts generated by alternative splicing of the BAX gene were differentially expressed in tumor and normal tissues,and ovarian cancer patients with less retention of the BAX exon 2 had poorer prognosis.Subsequently,we clarified the relationship between PQBP1 and mitochondrial apoptosis.In PQBP1 knockdown cell lines,the proportion of apoptotic cells increased,ROS levels increased,mitochondrial number decreased,mitochondrial membrane potential decreased,and the mitochondrial maximum oxygen consumption rate decreased.Overexpression of PQBP 1 can rescue the apoptosis of ovarian cancer cells and the decrease in cell proliferation caused by BAX.In conclusion,PQBP1 inhibits apoptosis and promotes malignant progression in ovarian cancer by regulating splicing and expression of BAX.Chapter Ⅲ:Antisense oligonucleotides targeting BAX exon 2 in treatment of ovarian cancer.We used the PQBP1 binding motifs reported in Chapter Ⅱ to design three thiophosphate bond-modified antisense oligonucleotides(ASOs)near BAX exon 2 for the treatment of tumor cells.Among the three ASOs,ASO1 can alter the BAX exon 2 splicing mode and induce apoptosis,and the killing effect of ASO1 on tumor cells was also demonstrated in vivo by animal experiments.In summary,we studied the role and mechanism of the splicing factor PQBP1 in ovarian cancer.The results showed that PQBP1 was highly expressed in ovarian cancer and correlated with poor prognosis.PQBP1 promoted tumor cell proliferation and inhibited tumor cell apoptosis.PQBP1 mainly binds to exons and the splicing site of precursor mRNA,and its ability to regulate expression of downstream target genes is significantly related to the abundance and location of binding sites.PQBP1 inhibits the apoptosis of ovarian cancer cells by regulating skipping of BAX exon 2.Antisense oligonucleotides targeting BAX exon 2 splicing can inhibit proliferation and induce apoptosis of ovarian cancer cells.This study provides a new target,PQBP 1,for early diagnosis of ovarian cancer and further discusses the mechanism of abnormal splicing in malignant tumors.Antisense oligonucleotide drugs designed for changing the BAX splicing pattern have small molecular weights and were conveniently synthesized with good application prospects in the treatment of cancer.