sortilin抑制ABCA1介导胆固醇流出的致动脉粥样硬化机制及miR-125a靶向调控作用研究

Latest studies reveal a close association between sortilin and plasma lipid level, atherosclerotic cardiovascular risk, while the underlying mechanism is unknown. ATP binding cassette transporter A1 (ABCA1) is a critical protein that mediates macrophage cholesterol efflux and prevents atherogenesis. Our pilot experiments confirmed sortilin decreases macrophage ABCA1 level, leads to the intracellular lipid accumulation and sortilin 3’UTR contains target binding sites for miR-125a. Therefore, we speculate that sortilin promotes lysosomal degradation of ABCA1 protein to downregulate its level, causing the obstruction of cholesterol efflux from macrophage and the development of atherosclerosis; and miR-125a directedly inhibits sortilin expression and elevates ABCA1 protein level. In this project, we first silence or over-express macrophage sortilin or miR-125a to detect sortilin expression, the alteration of ABCA1 degradation and level, and intracellular cholesterol efflux and lipid content; then we explore the effects of overexpression and silence of sortilin or miR-125a on the efficiency of reverse cholesterol transport, aortic ABCA1 protein level and atherosclerotic plaque area, and blood lipid profile in LDLr knockout mice. The project is hopeful to illuminate the effect and underlying mechanism of sortillin on ABCA1-mediated cholesterol efflux and atherogenesis, and the target regulation of miR-125a as well.

sortilin与血脂水平及动脉粥样硬化（As）性疾病密切相关，但机制未明。ABCA1是介导胞内胆固醇流出的关键蛋白，抗As。前期发现sortilin降低巨噬细胞ABCA1蛋白水平，促进胞内脂质蓄积，且sortilin 3’UTR含miR-125a靶向结合位点。据此推测sortilin促进ABCA1蛋白降解下调其表达水平，抑制巨噬细胞内脂质流出和促As；miR-125a靶向沉默sortilin上调ABCA1表达，抗As。本项目拟沉默或过表达巨噬细胞sortilin、miR-125a，检测sortilin表达、ABCA1蛋白降解及水平、胆固醇流出、胞内脂质含量；沉默或过表达LDLr敲除小鼠sortilin、miR-125a，观察对RCT效率、主动脉ABCA1表达、血脂、As斑块的影响。本项目的完成有望阐明sortilin抑制ABCA1介导胆固醇流出的致As机制及miR-125a靶向调控作用。

动脉粥样硬化（AS）早期病理变化是以动脉内膜下脂质沉积和泡沫细胞形成为主要特征，其中巨噬细胞内胆固醇蓄积在泡沫细胞形成及AS发展进程中起着重要作用。本项目以巨噬细胞脂质代谢为切入点，探讨了脂质代谢新调节因子分拣蛋白sortilin，是否促进三磷酸腺苷结合盒转运体A1（ABCA1）蛋白经溶酶体降解，下调巨噬细胞ABCA1蛋白水平而抑制胞内胆固醇流出，并进一步探讨miR-125a是否靶向沉默sortilin，抑制其介导ABCA1蛋白降解，促进胞内胆固醇流出，抑制泡沫细胞形成和As进展。结果发现，过表达sortilin减少荷脂巨噬细胞胆固醇流出，并增加THP-1巨噬细胞内脂质蓄积。免疫沉淀发现sortilin能与ABCA1结合，且过表达sortilin下调细胞内ABCA1蛋白水平，但对其mRNA水平无显著影响；当同时加入溶酶体抑制剂氯喹处理后，荷脂巨噬细胞ABCA1蛋白水平明显上调，而当加入蛋白酶体抑制剂MG132后ABCA1蛋白水平无明显改变。使用shRNA沉默sortilin表达后，ABCA1蛋白水平及其介导的胆固醇流出量明显增加。高表达sortilin的高脂饲喂LDLR−/−小鼠主动脉脂质沉积和斑块面积增加，并且斑块内ABCA1表达减少，而同时在体内高表达ABCA1或注射溶酶体抑制剂氯喹可逆转这一情况。生信分析表明miRNA-125a与SORT1 mRNA 3'UTR之间存在较保守结合位点，荧光素酶报告基因显示miRNA-125a可在细胞内作用于SORT1 mRNA 3'UTR；荷脂THP-1巨噬细胞转染miRNA-125a mimic后显示sortilin表达下调，并促进胞内胆固醇流出，减少脂滴数目和大小，减轻细胞脂质蓄积。高脂饲喂LDLR−/−小鼠体内高表达miRNA-125a，可下调主动脉sortilin水平，降低血脂水平，减少主动脉斑块面积和管壁脂质沉积，而转染miRNA-125a ASO则出现相反情况。以上结果表明，sortilin能够抑制ABCA1蛋白表达和胆固醇流出，促进巨噬细胞泡沫化和AS进展，而miRNA-125a沉默荷脂巨噬细胞sortilin表达，抑制细胞内胆固醇蓄积，抗AS。本项目的研究工作，从脂质代谢角度阐述了sortilin致AS作用新机制及sortilin受控新途径，为探讨AS发病机理、开发AS防治策略提供新实验依据和潜在靶标。