SIRT1介导组蛋白乙酰化在同型半胱氨酸致动脉粥样硬化中的作用及特异性miRNAs调控机制

Atherosclerosis (As) is a systemic disease which is characterized by lipid metabolism disorder. Previous research has shown that hypermethylation and hypomethylation were co-exist in the process of Hcy induced As,which indicates the existence of deeper regulatory mechanism. Histone acetylation regulates genetic transcription coordinate with DNA methylation, however, it has not been reported that whether Hcy causes As through abnormal histone acetylation and miRNAs is an important way in regulating gene expression；Therefore, this project intends to replicate the model of HHcy-induced As, detecting the changes of histone acetylation with western blot to clarify the role of H3 and H4 acetylation in As; detecting the expression of HDACs, HATs, and SIRT1 by qRT-PCR. Constructing the over-expressed and suppressive vectors of SIRT1 to clarify the mechanism of histone acetylation and determine whether SIRT1 is the key target gene. Microarray is used to screen Hcy specific miRNAs, and observe its effect on histone acetylation after transfecting the over-expressed and suppressive vectors of the specific miRNAs. Constructing the recombinant plasmid carrying SIRT1 and mutant luciferase reporter plasmid and transfect into related cells to discuss the mechanism of specific miRNAs target regulating SIRT1 and identify the key targets, to provide a theoretical basis for the targeted therapy of As.

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，提示存在更深层次机制；组蛋白乙酰化具有协同DNA甲基化调控基因转录的作用，但Hcy是否通过组蛋白乙酰化异常致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，免疫印迹检测组蛋白乙酰化的改变，阐明H3和H4乙酰化在As中的作用；定量PCR等检测HDACs、HATs及SIRT1的表达，构建针对SIRT1的过表达和抑制表达载体转染，阐明组蛋白乙酰化的机制并确定SIRT1是关键靶基因；运用微阵列技术筛选Hcy特异性miRNAs，转染miRNAs抑制物和过表达载体，观察其对组蛋白乙酰化的影响；构建携载SIRT1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SIRT1的机制，确定关键靶点，为As靶向治疗提供理论依据。

目的 探讨组蛋白H3K9乙酰化在同型半胱氨酸（homocysteine，Hcy）引起动脉粥样硬化（Atherosclerosis，AS）中的作用及机制，明确组蛋白去乙酰化酶1（histonedeacetylase 1，HDAC1）是调控H3K9乙酰化的重要酶，确定HDAC1对THP-1单核源性泡沫细胞脂质聚集的影响；阐明miR-34a对HDAC1的调控机制，旨在揭示miR-34a经HDAC1调节H3K9乙酰化促进Hcy引起AS的机制。.结果.1.小鼠主动脉根部冰冻切片HE和油红O染色结果显示，模型组（高蛋氨酸组）小鼠主动脉出现明显粥样斑块，高蛋氨酸组小鼠的血清Hcy水平明显升高，斑块中脂质相关蛋白ADFP表达明显增多。.2.高蛋氨酸组小鼠主动脉及Hcy干预后的泡沫细胞当中HDAC1 mRNA及蛋白表达水平显著升高，而H3K9乙酰化的水平明显下降。.3.泡沫细胞过表达HDAC1后TC、FC和TG含量明显增加，同时，H3K9的乙酰化水平下调，且ADFP也明显增加，而干扰HDAC1之后，可缓解这一结果。.4.模型组小鼠主动脉和泡沫细胞中miR-34a表达明显降低；而在泡沫细胞中过表达miR-34a则可显著下调HDAC1的表达，增加H3K9乙酰化的水平，抑制泡沫细胞脂质沉积，抑制miR-34a可显著增加HDAC1的表达，下调H3K9乙酰化的水平，增加泡沫细胞脂质聚集水平。分别构建携载有野生型和突变型HDAC1 mRNA 3’UTR序列双荧光素酶载体，与miR-34a在泡沫细胞当中共表达，结果显示，转染了野生型HDAC1 mRNA 3’UTR的泡沫细胞中，其荧光素酶活性显著低于转染了突变序列的泡沫细胞。.结论：miR-34a能够通过靶向结合HDAC13’UTR从而降低HDAC1的表达水平，而Hcy可引起miR-34a的表达下调，导致HDAC1升高、H3K9乙酰化水平降低，最终加速Hcy致泡沫细胞脂质聚积。