KDM5C通过Hippo-YAP1通路调控人宫颈癌发生的初步机制

Preliminary mechanism of KDM5C regulating the occurrence of human cervical cancer through the Hippo-YAP1 pathway

目的探讨组蛋白去甲基化酶5C(KDM5C)通过Hippo-YAP1通路调控人宫颈癌发生的分子机制。方法采用稳转过表达KDM5C蛋白的CaSki细胞系,应用转录组测序(RNA-Seq)技术进行全转录组测序分析差异表达基因,并对差异基因进行GO分析、KEGG分析和蛋白互作网络分析。随后用siRNA敲低KDM5C基因,并通过RT-qPCR和Western blotting等反向验证关键受调控基因Yes相关蛋白1(YAP1)的表达变化。对过表达KDM5C蛋白的CaSki细胞系通过染色质免疫共沉淀测序(ChIP-Seq)技术和ChIP-qPCR方法分析KDM5C蛋白对YAP1基因启动子区间甲基化状态的影响。结果RNA-Seq分析显示,过表达KDM5C蛋白导致356个mRNA表达明显上调和335个mRNA表达明显下调(P<0.05);GO富集分析显示,KDM5C蛋白主要参与机体各种生物发育过程;KEGG富集分析显示,KDM5C蛋白主要参与黏着斑连接、甾类激素生物合成、Hippo-YAP1信号通路、FoxO信号通路、凋亡和感染等过程。RT-qPCR分析结果显示,敲低基因KDM5C可上调YAP1基因的表达;Western blotting结果也显示,降低KDM5C蛋白的表达可同时上调YAP1和磷酸化YAP1的表达水平(P<0.05)。ChIP-Seq分析显示,过表达KDM5C蛋白可明显升高YAP1基因启动子区间的H3K4me1水平、并降低H3K4me3水平(P<0.05),ChIP-qPCR分析也验证了这种表达变化(P<0.05)。结论KDM5C蛋白可调控宫颈肿瘤细胞YAP1基因启动子的组蛋白H3K4me1/me3甲基化水平,进而影响YAP1基因的转录。YAP1蛋白作为Hippo-YAP1通路的核心因子,其表达改变直接影响细胞的黏附、增殖和凋亡等过程,进而参与宫颈癌的发生发展。

Objective To investigate the molecular mechanism of histone demethylase 5C(KDM5C)regulating human cervical carcinogenesis through Hippo-YAP1 pathway.Methods Using CaSki cell lines stably overexpressing KDM5C protein,whole transcriptome sequencing was performed by RNA-Seq technique,and differentially expressed genes were analyzed,and then GO analysis,KEGG analysis and protein interaction network analysis were performed on these differential genes.After that,siRNA knocked down KDM5C and reversely verified the expression changes of key regulated gene Yes associate protein 1(YAP1)by RTqPCR and Western blotting.Meanwhile,in CaSki cell lines,the effect of KDM5C protein overexpression on the methylation status of YAP1 gene promoter region was analyzed by chromatin immunoprecipitation sequencing(ChIP-Seq)and ChIP-qPCR methods.Results RNA-Seq analysis showed that overexpression of KDM5C significantly up-regulated expressions of 356 mRNAs and downregulated 335 mRNAs expressions(P<0.05).GO enrichment analysis showed that KDM5C protein was mainly involved in various biological development processes of the body.KEGG enrichment analysis showed that KDM5C protein was mainly involved in focal adhesion,steroid hormone biosynthesis,Hippo-YAP1 pathway,FoxO pathway,apoptosis and infection.Further RT-qPCR analysis showed that knockdown of KDM5C with gene specific siRNAs could up-regulate the expression of YAP1,and Western blotting results also confirmed that reduction the expression of KDM5C protein could up-regulate the levels of YAP1 and phosphorylated YAP1 simultaneously(P<0.05).ChIP-Seq analysis showed that KDM5C overexpression cell line could significantly increase the H3K4me1 level and decrease the H3K4me3 level in the promoter interval of YAP1 gene compared with the control cell line,and this expression change was also verified by subsequent ChIP-qPCR(P<0.05).Conclusions The KDM5C protein regulates the methylation level of the histone H3K4me1/me3 in the YAP1 gene promoter of cervical cancer cells,thereby affecting the transcription of the YAP1 gene.As a core factor in Hippo-YAP1 pathway,the expression of YAP1 protein directly affects cell adhesion,proliferation,and apoptosis,thereby participating in the occurrence and development of cervical cancer.