同型半胱氨酸经特异性lncRNA调控泡沫细胞自噬水平降低的表观遗传学机制研究

Autophagy is involved in the regulation of atherosclerosis (As) induced by homocysteine (Hcy), but the mechanism remains unclear yet. Previous studies found that DNA methylation regulate by DNMT1 is an important mechanism in Hcy induced As, and lncRNA participate in the regulation of autophagy in the form of epigenetics. Moreover, transcription factor Sp1 has a synergistic effect in this process, so it is speculated that Sp1 recruit DNMT1 to mediate DNA methylation of lncRNA promoter region to reduce the level of autophagy of foam cells is an important mechanism in atherosclerosis induced by Hcy. In order to verify this hypothesis, we will screen and identificate the specific lncRNA(lncAPF) of in Hcy-induced As, construct the lncAPF over-expression and the interference vectors. Transfect vectors into cells and perform vertification, so as to clarify the effect of lncAPF in the regulation of the decreased autophagy levels of foam cells. Analyse the lncAPF and its DNA methylation levels in its promoter region by the channels of MeDIP, clarifying the role and epigenetic alteration of lncAPF in foam cell autophagy. Using Electrophoretic Mobility Shift Assay ( EMSA) and Pull-down Assay, we aim to investigate the binding capacity and sites between Sp1 and DNMT1, revealing the mechanism of Sp1 which recruit DNMT1 to regulate the lncRNA promoter region DNA methylation leading to autophagy. In this way, we can explore the key targetsof As and provide a theoretical basis for the prevention and treatment of As.

自噬参与了同型半胱氨酸(Hcy)引起动脉粥样硬化(As)的调控，但机制未清。前期研究发现DNA甲基化是Hcy致病的重要机制，而lncRNA可经表观遗传学修饰调控自噬，且转录因子Sp1具有协同作用，故推测：Sp1募集DNMT1介导lncRNA启动子区DNA甲基化调控泡沫细胞自噬下降是Hcy引起As的重要机制。为验证该假说，拟筛选和确定Hcy引起As中特异性lncRNA(lncAPF)，分别构建其过表达或沉默载体并转染细胞予以验证，明确lncAPF在调控细胞自噬水平降低中的作用；MeDIP等分析细胞中lncAPF及其启动子区DNA甲基化状态，阐明lncAPF及其表观遗传学改变在细胞自噬中的作用；利用凝胶迁移电泳和CHIP等明确Sp1与DNMT1的结合能力和位点，揭示Sp1募集DNMT1调控lncRNA启动子区DNA甲基化引起自噬水平降低的机制，探寻关键靶点，为防治As提供理论依据。

背景：自噬参与了同型半胱氨酸(Hcy)引起动脉粥样硬化(As)的调控，但机制未清。前期研究发现DNA甲基化是Hcy致病的重要机制，而lncRNA可经表观遗传学修饰调控自噬，但Hcy是否及如何通过控调控lncRNA启动子区DNA甲基化起介导As的发生未见报道。.内容：通过复制ApoE-/-鼠高Hcy血症As动物模型，RNA-Seq等技术筛选并确定Hcy引起泡沫细胞中特异性lncRNA(提示为lncARF)；体外敲除lncRNA后检测泡沫细胞自噬水平，明确lncRNA在Hcy引起泡沫细胞自噬中的作用；生物信息学分析预测对lncARF的功能性靶蛋白(提示为LRP12)，RIP及RNA pulldown进一步验证lncARF与LRP12的结合；双荧光素酶报告系统和CHIP预测并分析转录因子(NKX2.5)与lncARF启动子结合情况；Massarray分析lncARF启动子区甲基化水平，并筛选其DNA甲基转移酶，明确lncARF启动子区DNA甲基化对于泡沫细胞自噬水平的影响。.结果：首先，RNA-Seq结果显示LncARF是Hcy引起泡沫细胞自噬水平降低中的特异性lncRNA，LRP12是其功能性靶蛋白；敲除LncARF和LRP12后自噬水平显著升高；进一步验证发现lncARF调控LRP12的表达且与LRP12相互作用；其次，生物信息学分析发现，转录因子NKX2.5与lncARF存在多个结合位点，且正向调控lncARF的表达；最后，甲基化分析显示Hcy干预后lncARF启动子区甲基化水平显著升高，DNMT1是其关键酶调控，过表达DNMT1后泡沫细胞自噬水平升高。.该项目已见刊发表SCI论文1篇，审稿中SCI论文1篇；培养青年教师2人、博士研究生1人、硕士研究生3人。.意义：本课题的实施将有利于阐明Hcy引起As的分子机制，确定关键靶点，为As的靶向治疗提供新的干预途径。