miR-200a通过调节PTEN影响小鼠肝细胞中AKT/GSK信号通路活性

摘要

目的 肝胰岛素抵抗能够导致严重的糖脂代谢紊乱,同2型糖尿病的发病密切相关.miR-200a属于miR-200家族,广泛表达于各个组织中,在许多癌症细胞中高表达.此研究探讨miR-200a通过下游靶基因PTEN调节AKT/GSK信号通路活性的机制,从microRNA角度阐明肝胰岛素抵抗的机制,为胰岛素抵抗的防治提供新的思路.方法 ① 脂质体在小鼠肝脏细胞株HEP1-6中转染miR-200a mimic和miR-200a inhibitor,用Real-time PCR检测细胞中miR-200a水平,并且检测AKT/GSK信号通路;② 用生物信息学方法预测miR-200a的下游靶基因;以双荧光素酶报告分析和Western印迹明确miR-200a的下游基因PTEN.③ 在HEP1-6细胞中共同转染miR-200a和si-PTEN,验证miR-200a通过PTEN调节AKT/GSK信号通路活性.结果 ① 用miR-200a mimic转染HEP1-6细胞,miR-200a水平升高,AKT/GSK信号通路活性增强;用miR-200a inhibitor转染HEP1-6细胞,miR-200a水平降低,AKT/GSK信号通路活性受到抑制;② 双荧光素酶报告分析和Western印迹结果表明,miR-200a能够直接同PTEN3′-UTR结合,抑制PTEN蛋白表达;③ 在HEP1-6细胞中沉默PTEN促进AKT/GSK信号通路活性;同时转染miR-200a和si-PTEN,能够逆转miR-200a inhibitor对AKT/GSK信号通路的抑制作用.结论 在HPE1-6细胞中miR-200a通过调节下游靶基因PTEN影响AKT/GSK信号通路活性.%Objective Hepatic insulin resistance leads to disorder of glucose and lipid metabolism,and it is closely associated with the pathogenesis of type 2 diabetes.miR-200a belongs to the miR-200 family and it extensively expresses in a great many of tissues.It has been reported that miR-200a is overexpressed in tumors.This study aimed to explore the effect of miR-200a on the activation of AKT/GSK pathway via targeting PTEN in an attempt to dissect the mechanism of insulin resistance and provide new ideas for the prevention and treatment of insulin resistance.Methods ① miR-200a mimic and miR-200a inhibitor were transfected into HEP1-6 cells.The expression of miR-200a was analyzed by real-time PCR.And the activation of AKT/GSK pathway was determined by Western blotting.② Bioinformatics' analysis was used to predict the target genes of miR-200a.Dual luciferase reporter assay and Western blotting were used to determine the effect of miR-200a on PTEN expression.③ miR-200a inhibitor and si-PTEN were co-transfected into HEP1-6 cells to confirm that miR-200a regulates the activation of AKT/GSK pathway via targeting PTEN.Results ① The level of miR-200a was increased and the AKT/GSK pathway activated in HEP1-6 cells transfected with miR-200a mimic,while the opposite findings were observed in HEP1-6 cells transfected with miR-200a inhibitor.② Dual luciferase assay and Western blotting showed that miR-200a could inhibit the expression of PTEN by directly binding to the 3′-UTR of PTEN.③ Down-regulation of PTEN reversed the effect of miR-200a inhibitor on the activation of AKT/GSK.Conclusion miR-200a regulates the activation of AKT/GSK pathway via targeting PTEN in HEP1-6 cells.