同型半胱氨酸经JAKs/STATs通路调控miR-200a组蛋白乙酰化致动脉粥样硬化的机制研究

Homocysteine (Hcy) is an independent risk factor of atherosclerosis (As). Its harmfulness is no less than hyperlipidemia but the mechanism is not clear now. It was confirmed that miRNAs can regulate gene expression in the post-transcription level, whose promoter region was affected by many factors. Meanwhile, acetylation of histone is also an important way involved with the regulation of gene expression. But it has not been reported that whether the occurrence of As induced by homocysteine (Hcy) result from abnormal histone acetylation of miRNAs promoter region. This project intends to replicate the Hcy models, so as to screen and identify the specific miRNAs of As caused by Hcy via gene chip; detect the changes of miR-200a and histone acetylation by the channels of qRT-PCR and ChIP; with the strategy of blocking and protein chip of JAKs and STATs, the mechanism of JAKs and STATs in the process of Hcy leading to As was revealed and the key targets were identified. Construct the truncated expression vector of STAT, then transfect truncated and full-length expression vectors into cells and analyze the alteration of miR-200a histone acetylation; study the effect of JAKs and SIRT1 and determined the structural domain, clarify the molecular mechanism of JAKs/STATs which regulate miR-200a histone acetylation. The aim of our research is to clarify the molecular mechanism of As caused by Hcy and to provide theoretical basis for its targeted therapy.

同型半胱氨酸(Hcy)是动脉粥样硬化(As)的独立危险因子，但机制未清。miRNAs在转录后水平调节基因表达，且其启动子区可受多种因素调控，而组蛋白乙酰化也是基因调控的重要方式，但Hcy经何种方式引起miRNAs启动子区组蛋白乙酰化致As未见报道。故采用基因芯片等筛选并确定Hcy引起As的特异性miRNAs(miR-200a)，ChIP分析miR-200a及其组蛋白乙酰化的变化，阐明组蛋白乙酰化在其中的作用；蛋白质芯片检测JAKs和STATs的变化并予以验证，明确Hcy引起As的关键亚型(JAK3/STAT3)；构建带标签的STAT3截短体载体片段，将其分别与SIRT1表达载体共转染，分析miR-200a启动子区组蛋白乙酰化变化，确定STAT3和SIRT1间的作用并确定结构域，揭示Hcy经JAK3/STAT3通路调控miR-200a组蛋白乙酰化的机制，为靶向治疗高Hcy血症提供理论依据。

动脉粥样硬化是心血管疾病的基础性病变，严重危害人群健康。研究表明，泡沫细胞累积是动脉粥样硬化发生的早期行为，对动脉粥样硬化发生发展及斑块稳定性具有重要影响。血浆同型半胱氨酸（Hcy）水平增高作为动脉粥样硬化的一项独立危险因素越来越受到人们的重视，但其具体致病机制及防治措施仍不清楚。microRNA（miRNA）是一组短的、具有组织特异性且高度保守的非编码RNA，在转录后水平调控基因表达，从而参与不同疾病病理生理过程。我们研究发现在高蛋氨酸饮食诱导的ApoE-/-鼠动脉粥样硬化模型中，高蛋氨酸饮食可显著升高血清Hcy水平，同时促进血清TC、TG、LDL-C升高，HDL-C下降，进而促进ApoE-/-鼠动脉粥样硬化斑块形成。研究发现在ApoE-/-鼠主动脉组织中miR-200a-3p表达水平升高，而miR-200a-5p表达水平变化不明显。同时，对体外培养的THP-1单核源性泡沫细胞给予Hcy干预后，miR-200a-3p表达水平也显著升高，明确了miR-200a-3p参与Hcy致动脉粥样硬化过程。基因芯片筛查发现Hcy引起泡沫细胞中上调的基因有1242个，下调的基因有493个。同时，本课题还探讨了miR-200a-3p表达改变的机制研究，相关结果在后续发表。为了进一步延伸该课题，我们还检测了在动脉粥样硬化形成过程中铁死亡的相关改变，结果显示，在高脂饮食组诱导的ApoE-/-鼠动脉粥样硬化模型中铁死亡相关因子p53的mRNA和蛋白表达明显升高，GPX4、SLC7A11 mRNA和蛋白表达明显降低；而在ox-LDL诱导的泡沫细胞中p53 mRNA和蛋白表达明显升高，GPX4、SLC7A11 mRNA和蛋白表达水平显著降低；脂质过氧化物ROS、MDA增多，Fe2+水平升高，GSH水平明显降低。最终明确了铁死亡参与高脂饮食诱导的ApoE-/-小鼠动脉粥样硬化的形成，且ox-LDL诱导泡沫细胞中脂质过氧化和铁死亡发生增加，提示泡沫细胞铁死亡可能与动脉粥样硬化的形成有关。