特异性lncRNA在同型半胱氨酸致动脉粥样硬化中表观遗传学机制研究

Homocysteine (Hcy) is an independent risk factor of atherosclerosis (As), which was based medical evidence, and its harmfulness is large, but the mechanism is not clear. We confirmed that DNA methylation is an significant mechanism of As in our previous studies, but what bothers us is that why hypermethylation and hypomethylation of different genes DNA could coexist under the same condition? At the same time, the study found that lncRNA can participate in the regulation of gene expression in coordinate with the other epigenetic modifications, and besides, the results of our pre-experiment indicated that lncRNA was involved in the regulation of As. So we speculate that Hcy may recuit epigenetic factors via lncRNA in order to mediate DNA methylation and histone methylation to modulate the functional target gene is an important mechanism of As. To verify this hypothesis, first and foremost, we ought to screen and identify the specific lncRNA (lncLSTR) of As induced by Hcy, and subsequently construct lncLSTR and its target gene nuclear factor IA (NFIA) adenovirus vector to inject into mice or transfect into foam cells respectively, and subsequently detect the vascular plaque area and lipid efflux in foam cells, so as to explore the role that lncLSTR and its functional target gene NFIA act in As. What’s more, to start with histones and DNA methylation in the promoter region of lncLSTR and NFIA through adopting the block-oriented strategy to explore their effect on the target gene after their balance model is broken. In this way, we will search for the key targets and provide new ideas in the prevention and treatment for As.

循证医学证据表明高同型半胱氨酸(Hcy)血症是动脉粥样硬化(As)的独立危险因子，危害性大，但机制未清。前期研究发现DNA甲基化是Hcy引起As的重要机制，但不解的是为何同条件下不同基因DNA高低甲基化并存？lncRNA可通过表观遗传学参与基因调控，且预实验提示其参与了As的调节，故推测：特异性lncRNA通过调控组蛋白和DNA甲基化介导靶基因表达是Hcy引起As的重要机制。为了验证该假说，拟筛选Hcy引起血管斑块中的特异性lncRNA(lncLSTR)并予以验证；构建lncLSTR及其功能性靶基因NFIA腺病毒载体分别注入小鼠和转染泡沫细胞，分析斑块面积和脂质流出情况，明确lncLSTR和NFIA在其中的作用；采用阻断为主的策略从lncLSTR及组蛋白和DNA甲基化等多向调控入手，探讨各自平衡模式被打破后对靶基因的影响，揭示lncLSTR调控NFIA的表观遗传学机制，为防治As提供思路。

背景：泡沫细胞脂质聚集是同型半胱氨酸(Hcy)致动脉粥样硬化(As)的重要机制。课题组前期证实lncRNA参与了Hcy引起泡沫细胞脂质聚集，但机制未清。DNA甲基化修饰是Hcy引起As的重要机制，但Hcy是否经特异性lncRNA调控DNA甲基化调节靶基因却未见报道。. 内容：通过复制ApoE-/-鼠高Hcy血症As动物模型，lncRNA-Seq等技术筛选并确定Hcy引起As血管斑块组织中特异性lncRNA；过表达或敲除lncRNA后检测泡沫细胞脂质沉积情况，明确lncRNA在Hcy引起泡沫细胞脂质沉积中的作用；应用在线软件对lncRNA的编码蛋白进行预测（提示为NFIA），Western blot及qRT-PCR检测小鼠血管组织与泡沫细胞中NFIA的表达改变；敲除或过表达NFIA后检测泡沫细胞脂质沉积情况；nMS-PCR检测Hcy作用下NFIA启动子区甲基化水平改变，并确定调控NFIA启动子区甲基化的关键酶；过表达或敲除lncRNA后，观察泡沫细胞中NFIA表达变化；同时，RIP检测lncRNA与NFIA的靶向结合关系，揭示lncRNA靶向调控NFIA是Hcy引起As的分子机制。. 结果：首先，lncRNA-Seq结果显示LncRNA LMRF是Hcy引起As中特异性lncRNA，敲除Lnc LMRF后泡沫细胞脂质沉积明显减弱；其次，生物信息学分析显示NFIA是Lnc LMRF的重要靶基因，且Hcy干预后泡沫细胞NFIA表达显著降低，而过表达NFIA脂质含量降低；进一步的甲基化分析显示Hcy干预后NFIA启动子区甲基化水平显著升高，DNMT1是调控其甲基化关键酶；最后，RIP结果显示NFIA与Lnc LMRF存在特异性结合并受其负向调控。. 该项目共发表SCI论文3篇，中文期刊2篇，出版专著1部，申请国家专利4个。. 意义：该项目将为Hcy引起As的治疗提供更稳定有效的靶点，有望发现Hcy发病机制的新规律，有望成为新一代As基因治疗手段。