H3K27me3和CFTR DNA甲基化在同型半胱氨酸致肝细胞凋亡中的相互作用机制研究

Homocysteine (Hcy) is not directly involved in lipid metabolism, but how can it destabilized the lipid transfer balance of vascular wall and ultimately lead to atherosclerosis (As) is not yet clear.Meanwhile,our earlier studies found that Hcy triggered the liver endoplasmic reticulum stress (ERs) and subsequent apoptosis of hepatic cell. DNA methylation is a major way to regulate the expressions of genes, and cystic fibrosis transmembrane conductance regulator (CFTR) is a key transmembrane factor which regulated the Ca2+,but whether Hcy through the H3K27me3 and CFTR methylation regulate ERs-mediated As has not been reported.This project is intended to replicate hyperhomocysteinemia (HHcy) animal models, CHIP was used to detected the changes of H3K27me3 and CFTR to confirm the role of them in cell apoptosis;With MeDIP-CHIP technology measured the DNA methylation levels of H3K27me3 and CFTR in the liver tissues, construction of gene interference H3K27me3 methylase and demethylase plasmid and then treated with antagonist, reversed the H3K27me3 methylation, starting from the bidirectional regulation mechanism of DNA and histone methylation, to explore the effect of H3K27me3 and CFTR DNA methylation interaction, and by which regulation mechanisms that Hcy-induced apoptosis, identified key targets, and provide experimental basis for targeted therapy of As.

同型半胱氨酸(Hcy)并不直接参与脂质代谢，何以能扰乱血管壁脂质转运平衡并导致动脉粥样硬化(As)尚未清楚。前期发现Hcy引起As时也触发了肝脏内质网应激(ERs)并引起细胞凋亡，CFTR是对Ca2+具有调节作用的关键跨膜因子，而表观遗传学修饰是基因表达调控的重要方式，那么Hcy是否通过H3K27me3和CFTR甲基化调控ERs介导As未见报道。本项目拟复制HHcy As模型，CHIP检测H3K27me3和CFTR的变化，明确H3K27me3和CFTR在细胞凋亡中的作用；MeDIP-CHIP检测肝组织中H3K27me3和CFTR甲基化改变，构建H3K27me3甲基化酶和去甲基化酶载体及拮抗剂干预，逆转H3K27me3甲基化，从DNA和组蛋白甲基化双向调控入手，阐明H3K27me3与CFTR DNA甲基化相互作用及在Hcy致细胞凋亡中的作用机制,确定关键靶基因,为As的靶向治疗提供实验依据。

本实验拟观察在同型半胱氨酸(Homocysteinemia,Hcy)致人肝细胞囊性纤维化跨膜转导调节因子(cystic fibrosis transmembrane conductance regulator,CFTR)表达改变过程中CFTR DNA甲基化及组蛋白甲基化发挥的调控作用及其机制。探讨同型半胱氨酸致AS的可能作用靶点。Hcy组较正常对照组肝细胞凋亡水平增加，Hcy+FV组较Hcy组肝细胞凋亡水平降低。Hcy组较正常对照组CFTR DNA甲基化水平升高，Hcy+FV组较Hcy组CFTR DNA甲基化水平降低；AZC组和EPZ005687组较正常对照组CFTR表达水平升高，Hcy+AZC组和Hcy+EPZ组较Hcy组CFTR表达水平升高，Hcy+AZC组较AZC组CFTR表达水平降低，Hcy+EPZ组较EPZ组CFTR表达水平降低。与正常对照组比较，Hcy组DNMT1表达水平升高，H3K27me3、EZH2表达水平升高，Hcy+FV组较Hcy组DNMT1表达水平降低，H3K27me3、EZH2表达水平降低。过表达和干扰DNMT1、EZH2后验证，病毒感染效率较高。过表达DNMT1后H3K27me3表达水平升高，干扰DNMT1后H3K27me3表达水平降低。过表达EZH2后CFTR DNA甲基化水平升高，干扰EZH2后CFTR DNA甲基化水平降低。与正常对照组相比，Hcy组、DNMT1+组和EZH2+组的H3K27me3与CFTR启动子区结合能力增强，DNMT1-组和EZH2-组的H3K27me3与CFTR启动子区结合能力减弱，而Hcy+FV组较Hcy组结合能力减弱。DNMT1和EZH2干扰病毒共感染后，CFTR表达水平增加。在同型半胱氨酸致CFTR表达降低的过程中，可能是由CFTR DNA甲基化和H3K27me3共同调控的，且两者之间存在相互调控关系。