miR-93-5p通过PI3K/AKT通路调控HepG2细胞自噬

miR-93-5p regulates autophagy of HepG2 cells through the PI3K/AKT pathway

目的:探究miR-93-5p对HepG2细胞增殖、自噬、葡萄糖消耗以及磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinases,PI3K)/蛋白激酶B(protein kinase B,AKT)通路的影响。方法:高浓度葡萄糖诱导构建胰岛素抵抗(insulin resistance,IR)细胞模型,设计合成miR-93-5p inhibitor和NC,结合自噬抑制剂3-MA,将细胞分为Control组、IR组、IR+inhibitor NC组、IR+inhibitor组、IR+3-MA+inhibitor组。CCK8法检测各组细胞增殖活力,试剂盒检测各组细胞葡萄糖消耗量和细胞糖原合成情况,Western-Blot法检测细胞自噬基因微管相关蛋白1轻链3(autophagy genes microtubule-associated protein 1 light chain 3,LC3)-I、LC3-II、肝细胞生长因子(hepatocyte growth factor,HGF)蛋白、PI3K/AKT通路蛋白(AKT、p-AKT)的表达。结果:与对照组相比,IR组细胞增殖活力、葡萄糖消耗量和细胞糖原合成量、HGF蛋白、LC3-II蛋白表达下降(P<0.01),LC3-I蛋白和p-AKT/AKT值表达均增加(P<0.01)。与IR组和IR+inhibitor NC组相比,IR+inhibitor组细胞增殖活力、葡萄糖消耗量和细胞糖原合成量、HGF蛋白、LC3-II蛋白表达增加(P<0.01),LC3-I蛋白和p-AKT/AKT值表达均下降(P<0.01)。与IR+inhibitor组相比,3-MA能逆转miR-93-5p inhibitor的作用。结论:miR-93-5p通过PI3K/AKT通路促进HepG2细胞自噬,进而抑制胰岛素抵抗,缓解糖尿病造成的机体损伤。

Objective:To investigate the effects of miR-93-5p on proliferation,autophagy,glucose consumption and phosphatidylinositol 3-kinases(PI3K)/protein kinase B(AKT)pathway of HepG2 cells.Methods:Insulin resistance cell model was induced by high glucose concentration,miR-93-5p inhibitor and NC were designed and synthesized,combined with autophagy inhibitor 3-MA.The cells were divided into Control group,IR group,IR+inhibitor NC group,IR+inhibitor group,IR+3-MA+inhibitor group.Cell proliferation activity was detected by CCK8 method,glucose consumption and glycogen synthesis were detected by kit,and the expression of autophagy genes microtubule-associated protein 1 light chain 3(LC3)-I,LC3-II,hepatocyte growth factor(HGF)protein and PI3K/AKT pathway proteins(AKT,p-AKT)were detected by Western-Blot method.Results:Compared with the control group,the cell proliferation activity,glucose consumption,glycogen synthesis and HGF protein,LC3-II protein expression in IR group were decreased(P<0.01),while the expressions of LC3-I protein and p-AKT/AKT value were increased(P<0.01).Compared with IR and IR+inhibitor NC group,cell proliferation activity,glucose consumption and glycogen synthesis,HGF protein,LC3-II protein expression were increased in IR+inhibitor group(P<0.01),LC3-I protein and p-AKT/AKT value expression were decreased in IR+inhibitor group(P<0.01).Compared with IR+inhibitor group,3-MA reversed the effect of miR-93-5p inhibitor.Conclusion:miR-93-5p promotes autophagy of HepG2 cells through PI3K/AKT pathway,thereby inhibiting insulin resistance and alleviating body damage caused by diabetes.