ciRs-0022098通过miR-342调控LIMP-2DNA甲基化在同型半胱氨酸引起动脉粥样硬化中的机制研究

Atherosclerosis (As) is a multifactor and complex disease which characterized by foam cells formation. The previous study of homocysteine (Hcy) caused As found that the coexistence of high and low DNA methylation level of different genes, suggesting a deeper mechanism. circRNA and miRNA belong to non coding RNA, which are similar in the mode of regulation, and lysosomal integral membrane protein 2(LIMP-2) is an important gene for regulating the transport of lipids. Therefore, this topic intends to establish the As model of hyperhomocysteinemia, to explicit the role of DNA methylation at LIMP-2 promoter region in Hcy-induced As and to determine that DNMT3a is the key regulatory enzyme; CiRs-0022098 and miR-342 were screened and identified as their specific circRNA and miRNA in Hcy-induced As; RNA-fluorescence in situ hybridization was used for analysis its expression and localization in the plaques; Using immune co-precipitation and biotin specific probe respectively precipitation or pull-down the compounds combined with ciRs-0022098and miR-342, to explicit the combination relations. The miR-342 luciferase reporter plasmid was constructed, and the ciRs-0022098 and miR-342 overexpression plasmid and/or co-transfected cells were used to elucidate the mechanism of the circRNA interacts with miRNA to regulate DNMT3a mediated LIMP-2 DNA methylation caused foam cells formation, which explore the key targets and provide the experimental basis for the prevention and treatment of As.

动脉粥样硬化(As)是以泡沫细胞形成为主要特征的多因素复杂性疾病。前期研究同型半胱氨酸(Hcy)引起As时发现不同基因DNA高低甲基化并存，提示存在更深层次的调控机制。环状RNA和miRNA同属非编码RNA，两者在调节方式上具有相似性，且LIMP-2是调控脂类转运的重要基因。故本课题复制高Hcy血症As模型，明确LIMP-2及其DNA甲基化在Hcy引起As中的作用并确定DNMT3a是其关键酶；筛选和确定ciRs-0022098和miR-342是其特异性环状RNA和miRNA；RNA荧光原位杂交等分析其表达并定位；生物素特异性探针和pull-down实验分别沉淀或下拉与两者结合的复合物并明确其结合关系；构建ciRs-0022098和miR-342过表达和干扰载体和/或共转染细胞，阐明两者相互作用调控DNMT3a介导LIMP-2表达引起泡沫细胞形成的机制，探寻关键靶点，为防治As提供实验依据。

背景：动脉粥样硬化(As)是以泡沫细胞形成为主要特征的多因素复杂性疾病。前期研究同型半胱氨酸(Hcy)引起As时发现不同基因DNA高低甲基化并存，提示存在更深层次的调控机制。环状RNA和miRNA同属非编码RNA，LIMP-2是调控脂类转运的重要基因。目前特异性环状RNA通过miRNA调控LIMP-2DNA甲基化在Hcy引起As中的作用尚不清楚。.内容：复制高Hcy血症As模型，免疫荧光、免疫印迹探讨LIMP-2在泡沫细胞脂质沉积中的作用；生物信息学分析LIMP-2启动子区CpG岛和GC含量，筛选并确定调控LIMP-2启动子区DNA甲基化改变的DNA甲基转移酶；RNA-seq高通量测序技术检测泡沫细胞中的circRNA，确定差异性表达的circRNA并检测其在泡沫细胞脂质沉积中的作用，生物信息学筛选出circRNA特异性靶向的miRNA，qRT-PCR检测miRNA的表达情况并进行功能验证，双荧光素酶实验证实circRNA和特异性miRNA存在结合位点，构建miRNA过表达和抑制载体并转染泡沫细胞，western blot检测过表达和抑制miRNA后泡沫细胞中脂质沉积情况。.结果：免疫荧光及Western-blot结果显示LIMP-2表达显著升高；生物信息学结果显示LIMP-2启动子区存在CpG岛(1235-1496)，Hcy干预后LIMP-2启动子区DNA甲基化水平显著降低且DNMT1表达降低，过表达DNMT1后LIMP-2启动子区DNA甲基化水平增高，脂质相关基因表达水平降低；RNA-seq高通量测序技术筛选Hcy干预的泡沫细胞中的特异性环状RNA为circPIAS1-5，RNA-FISH实验发现circPIAS1-5在细胞核质中均有表达，实验发现过表达circPIAS1-5后泡沫细胞中脂质沉积情况降低；miRanda软件预测显示circPIAS1-5与miR-219a-2-3p存在特异性结合位点，双荧光素酶报告进一步证实circPIAS1-5与miR-219a-2-3p存在特异性结合；油红O染色及Western-blot结果显示过表达miR-219a-2-3p后加速泡沫细胞脂质沉积，敲除miR-219a-2-3p抑制泡沫细胞脂质沉积，揭示circPIAS1-5与miR-219a-2-3p存在相互作用。.意义：该课题的实施有利于进一步研究As发生发展的分子机制。