特异性miRNAs经LSD1调节H3K9甲基化促同型半胱氨酸致动脉粥样硬化的研究

Atherosclerosis ( As ) is a multifactorial and complex disease with disorder of lipid metabolism. Previous studies on homocysteine (Hcy)-caused atherosclerosis found that different genes coexist with high and low methylation status under the same condition, which suggests that deepper regulation mechanisms may exist. The histone methylation has to coordinate the DNA methylation regulates gene transcription function. However, whether Hcy induce As through histone methylation has not been reported, but miRNAs is an important way for the regulation of gene expression.Therefore, this project plans to duplicate the HHcy As animal model, detect the changes of H3K9 in atheromatous plaque with CHIP and clarify its role in HHcy.Utilizate MeDIP-qPCR technology to detecte the levels of H3K9 methylation and LSD1 and RNA interference blocking strategy to identify key target., which to clarify the mechanism of H3K9 methylation in Hhcy. RNA interference blocking strategy to identify key target.Exploit microarray technology to screen HHcy specific miRNAs, the miRNAs inhibitor and overexpression vector transfected into foam cells, observe its effect on H3K9 methylation. Construct LSD1 recombinant plasmid and mutant luciferase reporter plasmid transfected into foam cells, which to explore the mechanisms of specific miRNAs targeted regulation LSD1 and Identify key targets. The purpose of this project is to ?provide theoretical basis for target therapy.

动脉粥样硬化(As)是以脂代谢紊乱为特征的全身性疾病，前期研究同型半胱氨酸(Hcy)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；组蛋白甲基化具有协同DNA甲基化调控基因转录的作用,但Hcy是否通过组蛋白甲基化异常引起As未见报道，而miRNAs是基因表达调控的重要方式，因此本课题拟复制HHcy As模型，CHIP法分析动脉斑块中H3K9的变化，明确其在HHcy中的作用；MeDIP-qPCR等方法检测斑块中H3K9甲基化和LSD1等调控因子的水平，采用RNA干扰等阻断策略确定关键靶点，阐明H3K9甲基化的作用机制；运用微阵列技术筛选HHcy特异性miRNAs，转染miRNAs抑制物和过表达载体，观察其对H3K9甲基化的影响；构建携载LSD1重组质粒及突变荧光素酶报告质粒并转染，探讨特异性miRNAs靶向调控LSD1的机制，确定关键靶点，为As靶向治疗提供理论依据。

动脉粥样硬化(As)是以脂代谢紊乱为特征的全身性疾病，前期研究同型半胱氨酸(Hcy)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；组蛋白甲基化具有协同DNA甲基化调控基因转录的作用,但Hcy是否通过组蛋白甲基化异常引起As未见报道，miRNAs是基因表达调控的重要方式。HHcy通过组蛋白甲基化和miRNA调控促进动脉粥样硬化的发生。本研究探讨了同型半胱氨酸(Hcy)对EZH2表达的影响，证实Hcy诱导的动脉粥样硬化是通过miR-92a调控的EZH2表达上调而引起的。高蛋氨酸饮食喂养16周后，APOE-/-小鼠主动脉EZH2表达上调、H3K27me3水平升高，而miR-92a表达下降。在泡沫细胞中过表达EZH2上调H3K27me3水平并加剧了泡沫细胞中总胆固醇和甘油三酯的积累。同时，上调miR-92a可降低泡沫细胞中EZH2的表达。这些数据表明，EZH2在高同型半胱氨酸介导脂质代谢紊乱中发挥重要作用，miR-92a可能是Hcy致动脉粥样硬化的一个新的治疗靶点。