MiR-27靶向调控LPL对动脉粥样硬化的影响

Accumulating evidence suggest that miR-27 may play important roles in atherosclerosis. However, many target genes of miR-27 have not been investigated in atherosclerosis thus sar. Interestingly, previous studies from our laboratory and others have shown that LPL might be a promising clinical approach for promoting clearance of triglyceride rich lipoproteins (TRLP), raising HDL and decreasing proin?ammatory roles in the treatment of atherosclerosis, suggesting that LPL may be a novel therapeutic target in the treatment of atherosclerosis. Of important, to search for a possible involvement of miR-27 in regulation of targets prediction information, we use many publicly available algorithms of databases. Our results have shown that the 3'UTR sequences of the LPL, which was predicted to bind to miR-27. Similarly, miR-27 speci?cally attenuates LPL expression in HAEC cells. Based on these studies, we hypothesized that miR-27 may promote atherosclerotic plaque development by directly targeting to LPL in vivo. In this research project, we will fuse a fragment of the human LPL 3'UTR harboring the predicted miR-27 target sequences from HAEC cells to a luciferase reporter plasmid for our experiments. Accordingly, we will observe the LPL expression and NF-кB pathway in HAEC cells through overexpression of miR-27 or knockdown of miR-27. Importantly, we will investigate the LPL expression and its tissue-speci?c activity, NF-кB pathway of arterial vessel, plasma levels of lipids, pro-in?ammatory cytokine secretion, TRLP clearance, the area of atherosclerotic plaque in apoE-/- mice by treated with miR-27. Furthermore, we will observe the area of atherosclerotic plaque in LPL-/- mice in order to elucidate that miR-27 may promote atherosclerotic plaque development by directly targeting to LPL in vivo. In the future, a better understanding of the mechanisms that underlie miR-27-mediated atherosclerosis,will enable miR-27-mediated therapy to open a new era in atherosclerosis care.

MiR-27促进动脉粥样硬化（As）的发生发展，但机制不清。课题组和国内外的研究显示水解甘油三酯的限速酶-LPL是防治As的重要靶点，预实验中我们发现miR-27具有靶向调控LPL的生物学基础，并具有沉默其表达的作用。因此，提出：miR-27靶向沉默LPL表达，引发脂质代谢紊乱和炎症效应，触发As发生的科学假说。项目拟采用荧光素报告基因分析miR-27与LPL的直接作用和结合位点；细胞实验观察miR-27对LPL表达及NF-κB信号通路的影响；动物实验观察miR-27对高脂喂养的apoE敲除鼠不同组织LPL表达、主动脉血管NF-κB信号通路、血脂、血浆炎症介质、富含甘油三酯脂蛋白清除效率及As斑块面积的影响；并观察对LPL敲除鼠As病变的影响，以明确miR-27通过LPL促进As发生发展的机制和作用。项目有望从新的视角阐明miR-27的致As机制，为其作为有益的治疗靶点提供新的实验基础。

MiR-27与动脉粥样硬化（As）性心血管疾病的发生发展密切相关，但是否参与调节As病理过程及其具体机制并不十分清楚。巨噬细胞源性脂蛋白酯酶（LPL）可通过“分子桥”作用促进对脂质的摄取，胞内脂质过度蓄积的同时增加炎症因子分泌，加速As病变的形成。课题组前期生物信息学分析发现miR-27具有靶向调控LPL的生物学基础，并通过预实验证实miR-27可以下调THP-1巨噬细胞LPL的表达。本项目通过探讨MiR-27调控LPL，影响巨噬细胞炎症因子分泌及脂质蓄积，研究MiR-27抗As作用及其可能机制，拟定以下实验方案进行研究：1.细胞实验观察miR-27对THP-1和RAW 264.7巨噬细胞炎症因子分泌及脂质蓄积的影响；2. 动物实验探讨miR-27对apoE-/-小鼠主动脉As病变形成的影响；3. 采用生物信息学技术和荧光素酶报告基因验证miR-27是否与LPL mRNA的3’UTR靶向结合并抑制该基因表达；体外及体内实验观测过表达或沉默LPL后，miR-27对炎症因子分泌及脂质蓄积的影响，并检测了炎症核转录因子NF-κB磷酸化水平及细胞表面清道夫受体表达情况，初步探讨miR-27的抗As机制以及巨噬细胞LPL在As发生发展中的作用，为防治As提供新的研究思路和治疗策略。