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

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.

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。

血管平滑肌细胞（VSMCs）增殖和迁移是动脉粥样硬化(As)的重要机制之一，高同型半胱氨酸(Hcy)血症是As的独立危险因素，而同型半胱氨酸致As时发现不同基因DNA高低甲基化并存，提示存在更深层次调控机制，miRNAs是基因表达调控的重要方式，因此本课题复制高Hcy血症VSMCs增生模型，qRT-PCR、western blot、MSP等方法检测VSMCs中PTEN、p53甲基化和DNMTs等调控因子的水平，采用RNA干扰等阻断策略确定关键酶；明确Hcy致VSMCs增殖改变中特异性miRNAs为miRNA-125b和miRNA-143，通过转染miRNA-125b和miRNA-143抑制物和过表达载体，观察其对VSMCs增殖和PTEN甲基化、p53甲基化的影响；构建携载DNMT3a和DNMT3b重组质粒及突变荧光素酶报告质粒并转染。结果发现VSMCs经Hcy干预后，DNMT3a和DNMT3b的表达增加，而miRNA-143和 miRNA-125b的表达被抑制，而miRNA-143和 miRNA-125b又可以靶向调控DNMT3a和DNMT3b的表达，即miRNAs和DNMTs具有相互调控作用。DNMT1的过表达增加PTEN甲基化水平并促进VSMCs增殖。此外，miRNA-143和miRNA-125b的上调降低了DNMT3a和DNMT3b的表达，并抑制了VSMCs增殖。最终我们的研究结果表明：1.Hcy促进平滑肌细胞增殖中DNMT1促进PTEN DNA甲基化改变；2.miR-125b靶向调控DNMT3b表达介导p53 DNA甲基化改变进而抑制Hcy对血管平滑肌细胞增殖作用；3.miR-143通过DNMT3a影响血管平滑肌细胞的增殖。