同型半胱氨酸经E2F1精氨酸甲基化调控内皮细胞凋亡和平滑肌细胞增殖共存的机制及干预靶位的研究

The coexistence of endothelial cells (ECs) apoptosis and vascular smooth muscle cells (VSMCs) proliferation is an important pathologic characteristic of atherosclerosis, and homocysteine (Hcy) is an independent risk factor for atherosclerosis, but it is remains unresolved that why the changes of two types of cells proliferous states are so different under the same condition. Arginine methylation is an important way of regulating gene transcription，and some studies have found that E2F1 is a common target gene of cell proliferation and apoptosis in recent years, but the mechanisms of E2F1 arginine methylation involved in the regulation of ECs apoptosis and VSMCs proliferation are not yet clear. Therefore, this subject intends to explore the role of E2F1 in Hcy-induced the coexistence of ECs apoptosis and VSMCs proliferation; To analyse the changes of E2F1 arginine residues methylation, screen and verify the specific arginine methyltransferase (PRMTs), the exploration of PRMT1 and PRMT5 modify adjacent three arginine residues(R109,R111,R113) of E2F1 competitively is the focus. To clarify its interaction domains and precise sites, reveal the mechanisms that Hcy regulate E2F1 arginine residues methylation by PRMTs; Using SELEX techniques to determine the intervention target of Hcy-induced the coexistence of ECs apoptosis and VSMCs proliferation. Our study will provide the theoretical basis for targeted therapy of As.

血管内皮细胞(ECs)凋亡和平滑肌细胞(VSMCs)增殖共存是动脉粥样硬化(As)的基本病理特征，同型半胱氨酸(Hcy)是As的独立危险因子，但何以同条件下两类细胞增殖态变化迥异至今无解。精氨酸甲基化是基因转录调控的重要方式，且近年来发现E2F1是细胞增殖和凋亡的共同靶基因，但E2F1及其精氨酸甲基化是否参与了调控ECs凋亡和VSMCs增殖及机制尚未清楚。因此本课题拟探讨E2F1在Hcy致ECs凋亡和VSMCs增殖共存中的作用；分析E2F1精氨酸残基甲基化的变化，筛选并确定特异性精氨酸甲基转移酶(PRMTs)，重点探寻PRMT1,5竞争性修饰E2F1精氨酸R109、R111、R113甲基化修饰的潜在差异，并明确其相互作用结构域和精确位点，阐明Hcy经PRMTs调控E2F1精氨酸甲基化的机制；运用SELEX技术确定Hcy致ECs凋亡和VSMCs增殖共存的干预靶位，为As靶向治疗提供理论依据。

背景：血管内皮细胞(ECs)凋亡和平滑肌细胞(VSMCs)增殖共存是动脉粥样硬化(As)的基本病理特征，同型半胱氨酸(Hcy)是As的独立危险因子，精氨酸甲基化是基因转录调控的重要方式，且近年来发现E2F1是细胞增殖和凋亡的共同靶基因，但E2F1及其精氨酸甲基化是否参与了调控ECs凋亡和VSMCs增殖及机制尚未清楚。.内容：本课题基于成功复制高同型半胱氨酸血症As模型的基础上，在整体和细胞水平利用免疫荧光、Edu染色、鬼笔环肽染色、细胞凋亡染色、免疫印迹、荧光PCR等探讨E2F1在Hcy致ECs凋亡和VSMCs增殖中的作用；分析E2F1精氨酸残基甲基化的变化，筛选并确定特异性精氨酸甲基转移酶(PRMTs)，探寻PRMT1,5竞争性修饰E2F1精氨酸R109、R111、R113甲基化修饰的潜在差异，并明确其相互作用结构域和精确位点，阐明Hcy经PRMTs调控E2F1精氨酸甲基化的机制；确定Hcy致ECs凋亡和VSMCs增殖的干预靶位。.结果：在动物水平和细胞水平检测到高蛋氨酸饮食或Hcy干预后E2F1的表达增加，ECs凋亡和VSMCs增殖增加；而干扰E2F1后能够降低ECs凋亡和VSMCs增殖水平；Hcy可以促进E2F1发生精氨酸甲基化改变；干扰PRMT1,5后，ECs凋亡和VSMCs增殖水平下降；生物信息学预测发现R109、R111、R113是E2F1的关键将氨酸甲基化位点；PRMT1,5重组质粒分别和E2F1精氨酸残基突变质粒共转后，免疫沉淀检(IP)测发现R109、R111/113分别为PRMT1和PRMT5的精氨酸甲基化修饰位点。.意义：该课题的实施有利于进一步研究动脉粥样硬化发生发展的分子机制，从翻译后修饰的角度着手，探寻关键靶点，为动脉粥样硬化的防治提供更多研究方向和资料。