miR-661靶向SCARB1调控冠状动脉内皮细胞胞吞LDL的机制研究

Mechanism of miR-661 targeting SCARB1 to regulate endocytosis of LDL in coronary artery endothelial cells

目的探讨miRNA如何介导清除剂受体B类成员1(SCARB1)调控冠状动脉(冠脉)内皮细胞胞吞低密度脂蛋白(LDL)。方法通过siRNA和过表达载体改变SCARB1在冠脉内皮细胞中的表达,检测冠脉内皮细胞胞吞天然LDL(nLDL)和循环氧化的LDL(oxLDL)的情况。将miRDB和HumanTargetScan预测的SCARB1潜在miRNA和已经发表的冠脉内皮细胞存在的miRNA取交集后筛选SCARB1潜在的miRNA。通过荧光素酶报告系统检测miRNA是否靶向SCARB1。结果敲低SCARB1后,冠脉内皮细胞胞内的nLDL和oxLDL均减少(P<0.05)。过表达SCARB1后,冠脉内皮细胞胞内的nLDL和oxLDL均升高(P<0.05)。将miRDB和HumanTargetScan数据库中预测的SCARB1的miRNA和已发表的冠脉内皮细胞的总miRNA表达谱做交集后,发现有6个miRNA分别是miR-328-5p,miR-661,miR-4306,miR-4650-5p,miR-5586-3p,miR-6885-5p。过表达相应的miRNA后,仅在过表达miR-661时冠脉内皮细胞中SCARB1的表达水平降低(P<0.05)。敲低miR-661后,冠脉内皮细胞中SCARB1的表达水平升高(P<0.05)。此外,miR-661靶向SCARB1的3端非编码区(P<0.05)。过表达miR-661后,冠脉内皮细胞nLDL和oxLDL的水平均降低(P<0.05),而敲低miR-661后,冠脉内皮细胞nLDL和oxLDL的水平均升高(P<0.05)。同时过表达miR-661和SCARB1后,冠脉内皮细胞nLDL和oxLDL的水平无明显变化(P>0.05)。结论miR-661通过靶向SCARB1 mRNA的3端非编码区降解SCARB1 mRNA后减少了SCARB1的翻译,最终抑制冠脉内皮细胞胞吞LDL。

Objective To investigate how miRNA mediates scavenger receptor B1(SCARB1)to regulate endocytosis of low-density lipoprotein(LDL)in coronary artery endothelial cells(CAEC).Methods The expression of SCARB1 was changed through siRNA and overexpression vectors in CAEC.The endocytosis of oxidized LDL(oxLDL)and natural LDL(nLDL)were detected in CAEC.The potential miRNA of SCARB1 predicted by miRDB and HumanTargetScan,and the published miRNA in CAEC were intersected to screen potential miRNAs of SCARB1.Whether miRNA targeted SCARB1 was detected by using luciferase reporter system.Results After knocking down SCARB1,intracellular nLDL and oxLDL decreased in CAEC(P<0.05).After SCARB1 had overexpression,intracellular nLDL and oxLDL increased in CAEC(P<0.05).After intersected miRNA of SCARB1with total expression profile of miRNA predicted in miRDB database and HumanTargetScan database,there were 6 miRNA found including miR-328-5p,miR-661,miR-4306,miR-4650-5p,miR-5586-3p and miR-6885-5p.The expression of SCARB1 decreased when only miR-661 had overexpression after overexpression of relative miRNA(P<0.05).After knocking down miR-661,expression of SCARB1 increased(P<0.05).In addition,miR-661 targeted 3-terminal noncoding region of SCARB1(P<0.05).After miR-661 had overexpression,levels of nLDL and oxLDL decreased(P<0.05),and after knocking down miR-661,levels of nLDL and oxLDL increased(P<0.05).After miR-661 and SCARB1 had onverexpression at the same time,levels of nLDL and oxLDL had no significant changes in CAEC(P>0.05).Conclusion MiR-661 reduces SCARB1 translation and finally inhibits endocytosis of LDL through targeting 3-terminal noncoding region of SCARB1 mRNA to degrade SCARB1 mRNA.