miR-488-3p靶向调控RAP1A在同型半胱氨酸介导肝细胞自噬的作用研究

Role of miR-488-3p targeting regulation of RAP1A in homocysteine-mediated hepatocyte autophagy

目的探讨微小RNA(miR-488-3p)通过靶向调控RAS癌基因家族成员RAP1A在同型半胱氨酸介导肝细胞自噬中的作用。方法体外常规培养人正常肝细胞(HL-7702),并给予100μmol/L Hcy干预24 h,采用Western blot检测肝细胞自噬相关蛋白LC3、p62和RAP1A的表达水平,qRT-PCR检测肝细胞中miR-488-3p和RAP1A的表达;转染miR-488-3p inhibitor和mimics后,qRT-PCR和Western blot分别检测miR-488-3p的转染效率及其对LC3和p62蛋白表达的影响;TargetScan预测miR-488-3p与RAP1A基因相关性,Western blot检测miR-488-3p下游潜在靶蛋白(RAP1A)的表达改变;Pearson相关系数对肝细胞中miR-488-3p表达水平与自噬相关蛋白水平进行相关性分析。结果与Control组相比,Hcy组肝细胞自噬相关蛋白LC3、RAP1A和miR-488-3p的表达水平升高(P<0.01),而p62表达明显降低(P<0.01);同时,转染miR-488-3p inhibitor和mimics后,与NC-inhibitor组比较,miR-488-3p inhibitor组中LC3和RAP1A蛋白的表达水平显著降低(P<0.01),p62表达明显升高(P<0.01);而与NC-mimics组相比,miR-488-3p mimics组中LC3和RAP1A蛋白表达水平明显增加(P<0.01),p62表达降低(P<0.01);进一步机制研究表明RAP1A是miR-488-3p的下游靶基因并受其正向调控。Pearson相关性分析发现,miR-488-3p的表达水平与LC3(r=0.9329,P=0.0002)蛋白表达呈正相关,而与p62(r=-0.8086,P=0.0083)表达则呈负相关。结论miR-488-3p在Hcy介导的肝细胞中高表达,可通过靶向调控RAP1A的表达促进肝细胞自噬。

Objective To investigate the role of the microRNA miR-488-3 p in homocysteine-mediated hepatocyte autophagy through targeted regulation of the RAS oncogene family member RAP1 A.Methods Human normal hepatocytes(HL-7702)were routinely cultured in vitro and administered 100μmol/L Hcy for 24 h.Western blot was used to detect expression levels of autophagy-related proteins LC3,p62 and RAP1 A in hepatocytes,whereas qRT-PCR was used to detect the expression of miR-488-3 p and RAP1 A in these cells.Following transfection of miR-488-3 p inhibitor and mimics,qRT-PCR and western blot were used to detect the transfection efficiency of miR-488-3 p and its effect on LC3 and p62 protein expression.TargetScan was used to predict the correlation between miR-488-3 p and RAP1 A genes,whereas western blotting was used to detect changes in the expression of RAP1 A,a potential downstream target protein of miR-488-3 p.Pearson’s correlation coefficient was used to evaluate potential correlations between expression levels of miR-488-3 p and autophagy-related proteins in hepatocytes.Results Compared with the control group,expression levels of autophagy-related proteins LC3,RAP1 A and miR-488-3 p in the Hcy group were increased(P<0.01),whereas expression of p62 was significantly decreased(P<0.01).Following administration of miR-488-3 p inhibitor and mimics,expression levels of LC3 and RAP1 A proteins in the miR-488-3 p inhibitor group were significantly decreased(P<0.01)and expression of p62 was significantly increased(P<0.01)compared with the NC-inhibitor group.Compared with the NC-mimics group,expression levels of LC3 and RAP1 A proteins in the miR-488-3 p mimics group were significantly increased(P<0.01),and expression of p62 was significantly decreased(P<0.01).Further mechanistic studies showed that RAP1 A is a downstream target gene of miR-488-3 p that is positively regulated by this miRNA.Pearson’s correlation analysis indicated that the expression level of miR-488-3 p was positively correlated with protein expression of LC3(r=0.9329,P=0.0002),but negatively correlated with p62 protein expression(r=-0.8086,P=0.0083).Conclusions miR-488-3 p is highly expressed in Hcy-mediated hepatocytes and can promote their autophagy by targeting expression of RAP1 A.