microRNA let-7c通过抑制PGC-1α/ABCA1途径促动脉粥样硬化及lncRNA Kcnq1ot1的调控机制研究

MiR-let-7c is involved in cholesterol metabolism, but its role in the accumulation of lipid in macrophages has not been elucidated. PGC-1α can promote ABCA1 expression to inhibit the accumulation of lipid in macrophages. Bioinformatics and preliminary experiments have found that miR-let-7c may inhibit peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) expression. The project will investigate more deeply on the direct effects of miR-let-7c on PGC-1α by report gene systems. In vitro experiments observe the effects of miR-let-7c on lipid accumulation and the expression of ABCA1.Then we explore the role of PGC-1α in regulation of ABCA1 expression by miR-let-7c. Furthermore, we evaluate the regulatory role of miR-let-7c on PGC-1α expression and plaque progression at systematic level. We found that miR-let-7c might be regulated by lncRNA Kcnq1ot1 through preliminary experiments. We will explore the upstream regulatory mechanisms of miR-let-7c by miR-let-7c sensor and RNA pull-down methods. In addition, the roles of miR-let-7c in lncRNA Kcnq1ot1 regulating accumulation of lipid in macrophages will be investigated. The completion of the project is expected to provide new targets and theoretical basis for the regulation of lipid accumulation of macrophages and the prevention and treatment of As in vivo.

miR-let-7c参与胆固醇代谢，但其在巨噬细胞脂质蓄积中的作用尚未阐明。PGC-1α可促进ABCA1表达抑制巨噬细胞脂质蓄积。生物信息学及预实验发现miR-let-7c可能靶向沉默PGC-1α。本项目拟用报告基因明确miR-let-7c对PGC-1α的直接作用，观察miR-let-7c对巨噬细胞脂质蓄积和ABCA1表达的影响，探讨PGC-1α在miR-let-7c调控ABCA1表达中的作用，整体上评价miR-let-7c对PGC-1α和As的作用。预实验发现miR-let-7c可能受lncRNA Kcnq1ot1的调控，我们将用miR-let-7c sensor和RNA pull-down等实验探讨miR-let-7c的受调控机制，以及miR-let-7c在lncRNA Kcnq1ot1调控细胞脂质蓄积中的作用。项目的完成有望为体内调控巨噬细胞脂质蓄积和As防治提供新的靶点和理论依据。

研究发现循环miR-let-7c的变化与脂质谱的变化显著相关，但其在细胞内脂质代谢中的作用尚不清楚。在本研究中，我们观察了miR-let-7c在动脉粥样硬化中的作用，并探索了潜在的分子机制。我们采用西方饮食饲喂ApoE-/-雄性小鼠20周，以构建动脉粥样硬化动物模型，同时经尾静脉注射特异性靶向CD68启动子的小鼠miR-let-7c基因抑制型AAV-9病毒（AAV9-CD68-miR-let-7c-inhibitor）完成相应处理。结果显示AAV9-CD68-miR-let-7c-inhibitor可减少ApoE-/-小鼠的斑块负荷。在体外实验中，miR-let-7c可通过抑制LXRa和ABCA1的表达抑制胆固醇外排，从而促进巨噬细胞脂质积累。通过生物信息学分析和荧光素酶报告基因检测，我们证实转录辅助活化因子a（PGC-1a）是miR-let-7c的下游靶基因。而且，PGC-1a表达在体内外均受miR-let-7c负向调控。此外，救济实验显示miR-let-7c对巨噬细胞脂质积累可被PGC-1a部分逆转。最后，通过生物信息学分析和荧光素酶报告基因检测，我们证实了miR-let-7c与LncRNA Kcnq1ot1靶向结合。而且，LncRNA Kcnq1ot1表达下调可抑制PGC-1a、LXRa和ABCA1的表达，从而促进巨噬细胞脂质积累。概而言之，我们的研究揭示了一种新的As发病机制，即miR-let-7c通过靶向PGC-1a抑制LXRa和ABCA1的表达，从而抑制胆固醇外排，促进巨噬细胞脂质积累，加速动脉粥样硬化发展。