lncRNA与miRNA相互作用调控TRPC6引起内质网应激在同型半胱氨酸致胰岛ß细胞凋亡的机制研究

Homocysteine (Hcy) is a major factor in induce type Ⅱ diabetes, but the mechanisms still unclear. Previous study has shown that miRNA involved in the pathogenic regulation of Hcy, but the regulation mechanism still need explore. LncRNA2 has structural consistency with miRNA in the, as well as similarity in the regulating mode, so the interaction between them becomes the focus of research. TRPC6 is a key gene in regulation of endoplasmic reticulum stress(ERs), thus it can be assumed that lncRNA interact with miRNAs to regulate TRPC6 is an important mechanism in Hcy-induced islet β cell apoptosis leading hyperglycemia. In order to verify this hypothesis, we aim to screen and identify that LOC105369458 and miR-3920 are the specific lncRNA and miRNAs in Hcy-induced hyperglycemia by the means of gene microarray. Performing immune co-precipitation and biotin specific probe respectively precipitate or drop-down the compounds combined with miR-3920 and LOC105369458, expliciting the combination relationships between them. Construct LOC105369458 and miR-3920 biosensor luciferase report plasmid, and transfect with both expression plasmid or transfecte respectively and analyze the change of the TRPC6, for revealing the interaction mechanisms by which regulate TRPC6 leading islet β cell apoptosis through ERs induced by Hcy, which provides an experimental basis for the prevention and treatment of Hcy.

胰岛β细胞凋亡是高同型半胱氨酸(Hcy)血症引起血糖升高的重要机制，但受何种因素及如何调控仍未清楚。lncRNA与miRNA参与了疾病的调控，且在结构上具有一致性，调节方式上具有相似性，因此探讨两者相互作用方式成为焦点；而TRPC6是调控内质网应激的重要基因，故推测：lncRNA与miRNA通过“海绵吸附”作用调控TRPC6经内质网应激引起胰岛β细胞凋亡是Hcy引起血糖升高的重要机制。为了验证该假说，基因测序筛选并确定LOC105369458和miR-3920是Hcy引起血糖升高的特异性lncRNA和miRNA；采用免疫共沉淀和生物素特异性探针分别沉淀或下拉与两者结合的复合物，明确其结合关系；构建miR-3920生物传感器荧光素酶报告质粒，并与两者过表达质粒和/或共转染细胞，分析TRPC6的变化，揭示两者相互作用调控TRPC6引起内质网应激致胰岛β细胞凋亡的机制，为糖尿病防治提供实验依据。

背景：胰岛β细胞凋亡是高同型半胱氨酸(Hcy)血症引起血糖升高的重要机制，lncRNA与miRNA在结构和调节方式上具有一致性和相似性，但两者调控胰岛β细胞凋亡在Hcy引起血糖异常中的作用未见报道。.内容：复制CBS+/-鼠高Hcy血症糖尿病动物模型，Western blot检测胰岛β细胞凋亡、TRPC6及ERs相关蛋白的表达情况；RNA测序等技术筛选并确定LOC105369458是Hcy引起胰岛β细胞中特异性lncRNA；转染LOC105369458慢病毒检测细胞凋亡及TRPC6蛋白表达情况；生物信息学分析预测miR-3920与LOC105369458相互作用，采用双荧光素酶报告检测两者结合情况，明确lncRNA与miR-3920相互作用调控TRPC6是Hcy引起胰岛β细胞凋亡的机制。.结果：凋亡相关蛋白bax与caspase-3蛋白表达升高，bcl-2表达降低，且TRPC6及ERs通路相关蛋白表达上升；敲除TRPC6后结果与之相反，表明TRPC6可通过ERs通路介导Hcy引起胰岛β细胞凋亡；测序结果显示LOC105369458是Hcy引起胰岛β细胞凋亡水平升高中的特异性lncRNA,可通过调控TRPC6介导Hcy引起胰岛β细胞凋亡；进一步研究发现LOC105369458与miR-3920相互作用，干扰LOC105369458发现miR-3920的表达降低，过表达miR-3920下调TRPC6的表达；干扰LOC105369458和miR-3920上调TRPC6的表达，证实lncRNA与miR-3920相互作用调控TRPC6是Hcy引起胰岛β细胞凋亡的机制。.该项目已见刊发表SCI论文5篇，审稿中SCI论文2篇；申请发明专利2项；培养青年教师2人、博士生1人、硕士生4人。.意义：本课题的实施将有利于阐明Hcy引起糖尿病的分子机制，确定关键靶点，为糖尿病的靶向治疗提供新的干预途径。