The Function And Potential Mechanism Of EZH2 On The Alternative Splicing In Prostate Cancer

Objective: Androgen-dependent prostate cancer cell lines(AD)and androgen-independent prostate cancer cell lines(AI)were selected and induced to explore the interaction between EZH2 and alternative splicing in prostate cancer cells,and to explore its function and mechanism which were independent of PRC2 complex.Methods: AD cell lines were obtained and cultured,and AI cell lines were constructed to simulate castration-resistant prostate cancer(CRPC)in clinic.After specific knockdown of EZH2 in cells by small interfering RNA(si RNA)and verification of knockdown efficiency by western blot and real-time quantitative fluorescence PCR(q PCR),a variety of cell samples were obtained.The interaction between EZH2 and chromatin in prostate cancer cells was detected and analyzed by chromatin immunoprecipitation sequencing(Ch IP-seq)and chromatin immunoprecipitation real-time fluorescence quantitative PCR(Ch IP-q PCR).RNA sequencing(RNA-seq),i RIP sequencing(i RIP-seq)and RIP-real-time fluorescent quantitative PCR(RIP-q PCR)were used to obtain and analyze the interaction between EZH2 and m RNA in prostate cancer.The interaction between EZH2 and histone was detected and analyzed by immunoprecipitation and peptide pull-down assays.Results: Through a series of Ch IP sequencing,RNA sequencing,i RIP sequencing and the corresponding real-time fluorescence quantitative PCR experiments,we obtained and compared the map of EZH2-chromatin and EZH2-RNA interactions,and tested the EZH2-chromatin contact model in prostate cancer cells.We found that EZH2 bound to the nascent transcripts in prostate cancer cells and had extensive binding effects not only to the precursor m RNA but also to spliced m RNA/lnc RNA.EZH2 binding signals in chromatin were positively correlated with gene expression levels and EZH2-RNA interaction sites.We also found that binding of EZH2 to transcriptional chromatin significantly increased its binding to H3K36me3 in a positive correlation.EZH2 was shown to bind to H3K36me3 in protein co-immunoprecipitation and peptide pull-down analysis.Chromatin deposition of H3K36me3 decreased after EZH2 expression was decreased.Peptide analysis also showed that the SET domain of EZH2 interacted with H3K36me3,suggesting that EZH2 might bind to chromatin with transcriptional activity through H3K36me3.In addition,EZH2 acted on chromatin to regulate cancer-related exon skipping,i.e.,EZH2 inhibited exon skipping,which was related to the binding of EZH2 to exons.Conclusions: EZH2 might have a function independent of PRC2 complex and might be associated with various cancer states by binding to the nascent transcripts of chromatin and RNAs,having effects on alternative splicing regulation,and possibly regulating other posttranscriptional regulation.These findings provided new mechanisms and ideas for the further study of EZH2.Inhibiting these binding and regulatory effects of EZH2 without interfering with its catalytic function in PRC could be a potential basis for the development of new therapeutic drugs and strategies.