miRNAs与DNA甲基转移酶1相互作用在同型半胱氨酸致血管平滑肌细胞增殖的分子机制

高同型半胱氨酸血症(HHcy)是动脉粥样硬化重要独立危险因子,前期研究HHcy时发现不同基因高低甲基化并存,提示存在更深层次调控机制。miRNAs是调控基因表达的重要方式,HHcy是否通过特异性miRNAs调控 DNA甲基化致VSMCs增殖不清楚。本项目拟复制HHcy致VSMCs增殖动物/细胞模型,检测VSMCs中PTEN 表达及其DNA甲基化和DNMT1等调控因子的变化,沉默/过表达DNMT1,阐明DNA甲基化的作用机制,确定关键靶点；微阵列技术筛选特异性miRNAs并予以验证，构建特异性miRNAs前体、抑制物和携载DNMT1靶向载体,明确VSMCs特异性miRNAs调控DNA甲基化的机制；用5-氮杂胞苷处理VSMCs,观察特异性miRNAs甲基化的变化，分析DNMTs负反馈调控miRNAs的作用机制。旨在阐明miRNAs与DNMT1相互调控的机制,为HHcy靶向治疗提供依据。

HHcy is an important and independant risk factor of atherosclerosis (As). Previous study has showed that hyper/hypo methylation is coexited in HHcy, which prompted that there might exist a deeper regulated mechanism. miRNAs is an important mechanism for the regulation of gene expression.However,whether HHcy induces VSMCs proliferation regulated by miRNA via DNA methylation or not is still not clear.This project intends to construct HHcy-induced proliferation of VSMCs animal and cell model.Then, detect PTEN expression and its DNA methylation and DNMT1 in VSMCs.Silence and overexpression DNMT1 to clarify the mechanism of DNA methylation. Microarray technology screen specific miRNAs and to be verified.The construction specificity miRNAs precursor, the inhibitor and take along carry the DNMT1 target vector to clarify the mechanism of miRNAs regulated DNA methylation. 5-azacytidine treatment VSMCs and analyse the change of specific miRNAs methylation.Observing the mechanism of DNMT1 negative feedback regulated miRNAs.The purpose of the reasearch is to reveal the mutual control mechanism between DNMTs and miRNAs and provide a theoretical basis for targetting therpy of As.