组蛋白H3K27me3介导JAK_(2)/STAT_(3)信号通路对足细胞损伤的调控机制

Histone H3K27me3 Mediates the Mechanism of JAK_(2)/STAT_(3) Signaling Pathway on Podocyte Damage

目的:探讨组蛋白H3K27me3介导JAK_(2)/STAT_(3)信号通路对足细胞损伤的调控机制。方法:体外培养小鼠肾足细胞MPC5,随机分为对照组及EZH_(2)-组。EZH_(2)-组小鼠肾足细胞给予EZH_(2)酶抑制剂(EPZ-6438)干预,对照组给予等量PBS培养。应用Western Blot法检测足细胞组蛋白H3K27me3、p-JAK_(2)、JAK_(2)、p-STAT_(3)、STAT_(3)蛋白水平;实时定量PCR法检测足细胞JAK_(2)、STAT_(3)mRNA水平;ChIP-chip确定组蛋白H3K27me3调控靶基因群,利用MACS2软件得出组间差异富集峰,进行基于GO、KEGG数据库的功能注释分类和通路富集分析。结果:与对照组相比,EZH_(2)-组足细胞H3K27me3蛋白表达显著下降(P<0.05),JAK_(2)/STAT_(3)信号通路相关基因及蛋白表达显著增加(P<0.05);GO富集分析发现靶基因Pigu基因呈现显著性富集,其参与JAK/STAT信号通路调控;KEGG富集分析发现靶基因参与调控信号通路主要集中在细胞溶质DNA感应途径、Rap1信号通路、Toll样受体信号通路、HIF-1信号通路和JAK/STAT信号通路,其中靶基因IL12rb1、IL5ra呈现显著性富集,其参与JAK/STAT信号通路调控。结论:组蛋白H3K27me3可能通过调控靶基因Pigu、IL12rb1、IL5ra参与JAK_(2)/STAT_(3)信号通路活化,导致足细胞损伤。

Objective:The present study aims to investigate the regulatory mechanism of histone H3K27me3 mediates the mechanism of JAK_(2)/STAT_(3)signaling pathway on podocyte damage.Methods:MPC5 mouse renal podocytes were cultured in vitro and randomly divided into control group and EZH_(2)-group.EZH_(2)-group were treated with EZH_(2)-enzyme inhibitor(EPZ-6438),control group were treated with PBS.The expression of histone H3K27me3,p-JAK_(2),JAK_(2),p-STAT_(3)and STAT_(3)protein was measured by Western Blot.The expression of JAK_(2)and STAT_(3)gene was measured by real-time PCR.ChIP-chip was used to determine histone H3K27me3 regulatory target gene group,MACS2 software was used to obtain differential enrichment peaks between the two groups and analyzed for functional annotation classification and pathway enrichment based on GO and KEGG databases.Results:Compared with the control group,the expression of histone H3K27me3 in EZH_(2)-group was decreased significantly(P<0.05);the expression of genes and proteins related to JAK_(2)/STAT_(3)signaling pathway was increased significantly(P<0.05);GO enrichment analysis found that Pigu gene was showed significant enrichment and was involved in the regulation of JAK/STAT signaling pathway;KEGG enrichment analysis showed that the involvement of target genes in the regulation signaling pathways was mainly concentrated in the cell solide DNA induction pathway,Rap1 signaling pathway,Toll-like receptor signaling pathway,HIF-1 signaling pathway and JAK/STAT signaling pathway,among which target genes IL12rb1 and IL5ra showed significant enrichment and were involved in the regulation of JAK/STAT signaling pathway.Conclusion:Histone H3K27me3 participates in the activation of JAK_(2)/STAT_(3)signaling pathway leading to podocyte damage by regulating target genes Pigu,IL12rb1 and IL5ra.