microRNA在绝经后胆固醇代谢紊乱中的作用及机制研究

The disturbance of cholesterol metabolism in postmenopausal women is related to the occurrence and development of cardiovascular disease (CVD). Therefore, nowadays, it is an important issue that clarifying the mechanism of disturbance of cholesterol metabolism and improving the state of metabolic disturbance after menopause to prevent CVD in postmenopausal women. The complement of estrogen can ameliorate the disturbance of cholesterol metabolism after menopause, meanwhile, it also increase the risk of female genital system tumors. Thus, it must be enforced to find effective advisable therapeutic targets. microRNA (miRNA) plays an important role in lipid metabolism. However, it is still unclear what role miRNA play in disturbance of cholesterol metabolism after menopause; therefore, this problem is regarded as a key breakthrough point and extended the following questions: which miRNA are involved in the process of disturbance of cholesterol metabolism after menopause? What are their target genes regulated by these miRNA? By what mechanism do the target genes regulated by miRNA participate in regulation of cholesterol metabolism after menopause? Based on these questions, we will study systematically effects and mechanisms of microRNA and their target genes on disturbance of cholesterol metabolism after menopause. Finally, with miRNA as therapeutic target, puting forward new prevention strategy to control disturbance of cholesterol metabolism after menopause, and then reducing the risk of CVD in postmenopausal women.

女性绝经后出现的胆固醇代谢紊乱与心血管疾病（CVD）的发生发展密切相关，因此明确绝经后胆固醇代谢紊乱的机制并改善这种紊乱状态，是当今世界女性CVD防治的重点。补充雌激素可改善女性绝经后胆固醇代谢紊乱，但同时也增加了生殖系统肿瘤的发病风险，故寻找有效且针对性强的治疗靶点势在必行。microRNA（miRNA）在脂类代谢中发挥了重要作用，但miRNA在女性绝经后胆固醇代谢紊乱中扮演何种角色尚不清楚，故本课题以此作为切入点，研究探讨以下问题：绝经后胆固醇代谢紊乱时哪些miRNA参与其中？这些miRNA调节的靶基因有哪些？靶基因又是通过何种机制参与调节绝经后胆固醇代谢紊乱的？针对以上问题，我们将全面系统的研究miRNA及其靶基因在绝经后胆固醇代谢紊乱中的作用及其机制。最终以miRNA作为靶点，提出绝经后胆固醇代谢紊乱的新防控策略，针对性防治女性绝经后胆固醇代谢紊乱，降低绝经后女性CVD的发病风险。

女性绝经后出现的胆固醇代谢紊乱与心血管疾病（CVD）的发生发展密切相关，因此明确绝经后胆固醇代谢紊乱的机制并改善这种紊乱状态，是当今世界女性CVD防治的重点microRNA（miRNA）在脂质代谢中发挥了重要作用，但其在女性绝经后胆固醇代谢紊乱中扮演何种角色尚不清楚。本项目通过体内及体外实验，寻找参与调节绝经后胆固醇代谢紊乱的主要miRNA及其所调节的靶基因，并明确其在绝经后胆固醇代谢紊乱中的作用和机制。本项目研究分为三个部分：.1. 绝经后胆固醇代谢紊乱过程中鼠肝脏组织miRNA表达谱的筛选。动物模型结果发现卵巢切除后小鼠雌激素水平显著下降，体重增加，普遍出现脂质代谢紊乱及脂质堆积现象。肝脏组织miRNA表达谱显示，与正常组相比，卵巢切除组小鼠肝组织中miRNA上调及下调2倍以上者分别为108种和111种。选取部分显著改变的miRNA，应用Real-time PCR及细胞实验进行验证，最终选取miR-203、miR-1b和miR-452作为候选miRNA进行进一步研究。.2. 筛选并验证miR-203、miR-1b和miR-452调节的靶基因。首先应用TargetScan、MICRORNA及RNAhybrid等数据库、软件等初筛出miR-203、miR-1b和miR-452可能参与胆固醇代谢调节的的靶基因Cyp7a1和Srebf2。同时，应用Western blot检测动物及细胞模型中Cyp7a1和Srebp2蛋白表达情况。最终通过luciferase双报告基因技术直接验证Cyp7a1及Srebf2分别为miR-203、miR-1b和miR-452调节的靶基因。.3. miRNA及其靶基因在绝经后胆固醇代谢紊乱过程中的调节作用机制研究。细胞水平，分别过表达或抑制miR-203、miR-1b和miR-452，其靶基因Cyp7a1和Srebp2蛋白表达相应降低或增高，随之细胞内胆固醇水平也发生改变。动物水平，去卵巢小鼠注入AMO-203后，其靶基因Cyp7a1表达升高，血清胆固醇水平有所下降，说明导入AMO-miRNA后能够改善绝经后小鼠胆固醇代谢紊乱。.综上所述，本项目系统研究了miRNA及其靶基因在绝经后胆固醇代谢紊乱中的作用及机制。最终以miRNA为靶点，提出绝经后胆固醇代谢紊乱的新防控策略，进而降低绝经后妇女心血管疾病的发病风险。