miR-206调控SIRT1/AMPK通路影响脂质代谢改善非酒精性脂肪肝的研究

MiR-206 Regulates Lipid Metabolism to Improve non-Alcoholic Fatty Liver Disease by The SIRT1/AMPK Pathway

目的:探讨miR-206对长链游离脂肪酸(FFA)诱导HepG2细胞建立的非酒精性脂肪肝(NAFLD)细胞模型的影响及其对沉默信息调节因子1(SIRT1)/AMPK通路的调控。方法:用1mM的FFA处理HepG2细胞24 h构建NAFLD细胞模型,根据转染物质的不同分为6组:Control组(无任何处理)、FFA组(构建NAFLD模型)、FFA+mimics NC组(转染mimics NC质粒)、FFA+mi R-206 mimics组(转染miR-206 mimics质粒)、FFA+Vector组(转染Vector质粒)、FFA+OE-SIRT1组(转染OE-SIRT1质粒)。油红O染色法检测细胞中脂质蓄积情况,酶联免疫吸附试验(ELISA)方法检测谷草转氨酶(AST)、丙氨酸氨基转移酶(ALT)及甘油三酯(TG)含量,荧光定量聚合酶链反应(qRT-PCR)检测mi R-206、SIRT1 mRNA表达量,Western blot法检测SIRT1、固醇调节元件结合蛋白1(SREBP1)、脂肪酸合成酶(FAS)、硬脂酰辅酶Al(SCD1)、乙酰CoA羧化酶1(ACC1)、白细胞分化抗原36(CD36)、AMP活化蛋白激酶(AMPK)及p-AMPK蛋白水平,TargetScan在线网站预测miR-206与SIRT1结合位点,双荧光素酶报告基因实验验证miR-206与SIRT1的靶向关系。结果:与Control组比较,FFA组细胞AST、ALT及TG含量明显升高,TG含量升高,miR-206、SIRT1蛋白及基因mRNA含量明显降低,SREBP1、FAS、SCD1、ACC1及CD36蛋白含量升高,p-AMPK/AMPK比值降低,差异具有统计学意义(P均<0.01)。与FFA+mimics NC组细胞比较,FFA+mi R-206 mimics组细胞内TG含量下降,SREBP1、FAS、SCD1、ACC1及CD36蛋白水平降低,SIRT1蛋白及基因m RNA水平升高,p-AMPK/AMPK比值升高,差异具有统计学意义(P均<0.01)。与FFA+Vector组细胞比较,FFA+OE-SIRT1组细胞内TG含量下降,SREBP1、FAS、SCD1、ACC1及CD36蛋白水平降低,p-AMPK/AMPK比值升高,差异具有统计学意义(P均<0.01)。TargetScan在线网站预测发现,miR-206与SIRT1野生型存在结合位点。SIRT1 WT+miR-206 mimics组细胞荧光素酶活性高于SIRT1 WT+mimics NC组,差异具有统计学意义(P<0.01)。结论:上调miR-206能够通过SIRT1/AMPK通路减轻NAFLD引起的肝细胞脂质代谢。

Objective:To investigate the effect of mi R-206 on the non alcoholic fatty liver(NAFLD)cell model induced by long chain free fatty acids(FFA)in HepG2 cells and its relationship with silent information regulatory factor 1(SIRT1)/AMPK pathway.Methods:HepG2 cells were treated with 1mM FFA for 24 hours to construct a NAFLD cell model.According to the different transfected substances,they were divided into 6 groups:Control group(without any treatment),FFA group(NAFLD model),FFA+mimics NC group(transfect mimics NC plasmid),FFA+miR-206 mimics group(transfect mi R-206 mimics plasmid),FFA+Vector group(transfect Vector plasmid),and FFA+OE-SIRT1 group(transfect OE-SIRT1 plasmid).Oil red O staining was used to detect lipid accumulation in cells.Enzyme-linked immunosorbent assay(ELISA)was used to detect the content of aspartate aminotransferase(AST),alanine aminotransferase(ALT),and triglycerides(TG).Fluorescence quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression of miR-206 and SIRT1 mRNA.Western blot was used to detect SIRT1,sterol regulatory element binding protein 1(SREBP1),fatty acid synthase(FAS),stearoyl coenzyme Al(SCD1),acetyl CoA carboxylase 1(ACC1),leukocyte differentiation antigen 36(CD36),AMP activated protein kinase(AMPK),and p-AMPK protein levels.TargetScan was used to predict the binding site of miR-206 to SIRT1.Dual luciferase reporter gene experiments was used to validate the targeting relationship between miR-206 and SIRT1.Results:Compared with the Control group,the FFA group showed a significant increase in AST,ALT,and TG content,an increase in TG content,a significant decrease in miR-206,SIRT1 protein and gene mRNA content,an increase in SREBP1,FAS,SCD1,ACC1,and CD36 protein content,and a decrease in p-AMPK/AMPK ratio(P<0.01).Compared with the FFA+mimics NC group cells,the FFA+miR-206 mimics group cells showed a decrease in intracellular TG content,a decrease in SREBP1,FAS,SCD1,ACC1,and CD36 protein levels,an increase in SIRT1 protein and gene m RNA levels,and an increase in p-AMPK/AMPK ratio,with statistically significant differences(P<0.01).Compared with the FFA+Vector group cells,the FFA+OE-SIRT1 group cells showed a decrease in TG content,a decrease in SREBP1,FAS,SCD1,ACC1,and CD36 protein levels,and an increase in p-AMPK/AMPK ratio(P<0.01).There was a binding site between miR-206and the SIRT1 wild-type.The cell luciferase activity in the SIRT1 WT+miR-206 mimics group was higher than that in the SIRT1WT+mimics NC group(P<0.01).Conclusion:Upregulation of miR-206 can alleviate NAFLD induced lipid metabolism in liver cells through the SIRT1/AMPK pathway.