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miRNA-26a抑制ox-LDL介导的HAECs凋亡作用的机制研究

         

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目的:探究miR‐26a在ox‐LDL介导内皮细胞HAECs凋亡中的作用及其调控机制。方法采用不同浓度的氧化低密度脂蛋白(ox‐LDL)在体外作用于HAECs细胞,噻唑蓝(MTT)和TUNEL染色检测ox‐LDL作用HAECs后细胞的活性与凋亡率,定量实时聚合酶链反应(qRT‐PCR)检测ox‐LDL作用HAECs后细胞中miR‐26a的表达水平。在HAECs中过表达miR‐26amimic,MTT和TUNEL染色检测ox‐LDL作用后细胞的活性和凋亡率。构建荧光素酶报告载体pMIR‐PTEN的3′UTR,利用荧光素酶活性检测鉴定miR‐26a的预测靶基因。qRT‐PCR和蛋白质印迹法(Westernblot)分别检测PTEN的mRNA和蛋白表达水平。结果ox‐LDL能够介导HAECs细胞的毒性死亡和细胞凋亡,并且降低了HAECs细胞中miR‐26a的表达水平。过表达miR‐26amimic能够抑制ox‐LDL作用HAECs后细胞的毒性和凋亡。转染miR‐26amimic显著抑制荧光素酶的活性(P<0.05)。转染miR‐26amimic显著下调HAECs细胞中PTEN的mRNA和蛋白表达水平(P<0.05)。结论miR‐26a能够抑制抑制ox‐LDL作用HAECs后细胞的毒性和凋亡,其可能的作用机制是下调了PTEN的表达。miR‐26a可能成为治疗凋亡相关的动脉粥样硬化的潜在靶点。%Objective To investigate the role of miR‐26a in ox‐LDL‐mediated apoptosis of HAECs in endothelial cells and its mechanism .Methods Various concentrations of ox‐LDL were added in HAECs culture .Cell cytotoxicity and apoptosis were moni‐tored by MTT and TUNEL assay ,and expression level of miR‐26a examined by qRT‐PCR .Overexpression of miR‐26a mimic in HAECs ,MTT and TUNEL staining were used to detect the activity and apoptosis of ox‐LDL .The 3′UTR of luciferase reporter vector pMIR‐PTEN was constructed and the predicted target gene of miR‐26a was identified by luciferase activity assay .QRT‐PCR and Western blot were used to detect the mRNA and protein expression of PTEN .Results ox‐LDL could mediate the toxic death and apoptosis of HAECs cells ,and decrease the expression level of miR‐26a in HAECs cells .Overexpression of miR‐26a mimic could inhibit the cytotoxicity and apoptosis of ox‐LDL cells after HAECs .Transfection of miR‐26a mimics significantly inhibited lu‐ciferase activity (P<0 .05) .The expression of mRNA and protein in HAECs cells was significantly down regulated by transfection of miR‐26a analog (P<0 .05) .Conclusion MiR‐26a can inhibit the cytotoxicity and apoptosis of ox‐LDL cells after HAECs inhibi‐tion ,and the possible mechanism of action is to down regulate the expression of PTEN .The study suggests that miR‐26a may be a potential target for the treatment of atherosclerosis related to apoptosis .

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