The Effect And Molecular Mechanism Of MicroRNA-22 On Hepatocyte Senescence Induced By High-Fat Conditions

Objective To investigate the mechanism of microRNA-22 on hepatocyte senescence in high-fat conditions,and to provide a potential target for effective intervention of non-alcoholic fatty liver disease.Methods(1)20 BALB/C mice were randomly divided into two groups,normal diet group and high fat diet group.The high fat diet group was fed with high fat diet(HFD)to induce non-alcoholic fatty liver disease(NAFLD).After 20 weeks,the mice body weight,liver weight and epididymis fat weight were weighed;Biochemical reagent kits were used to detect the liver triglyceride(TG)content;Hematoxylin eosin(H&E)staining was used to explore liver histopathology;The semi quantitative PCR was used to detect the mRNA expression levels of lipid metabolism related genes Lxr,Acsl,Acc1,Chrebp,and P21.(2)Senescence associated β-galactosidase staining(SA-β-Gal)was used to detect the senescence of hepatocytes;Then immunofluorescence staining was used to detect protein expression of P53 and sirt1;The real-time quantitative PCR was used to validate the relative expression of miR-22 and Sirt1;The BD cytometric bead array was used to detect the expression of IL2,IL4,IL6,IFNγ,TNF,IL17 A and IL10.(3)The MTT test was used to optimize the concentration of palmitic acid for the cellular senescence.β-galactosidase staining was used to observe the senescence after PA treatment;The real-time quantitative PCR was used to detect the relative expression of cell cycle and senescence related genes P53,P21,Cdk2,Cdk6,and the relative expression levels of miR-22,sirt1 and SASP inflammatory factors in BNL.CL2 cells exposed to PA.The immunofluorescence staining was used to detect protein expression of SIRT1,P53 and P21 in BNL.CL2 cells.(4)After miR-22 mimics was transiently transfected into BNL.CL2 cells,β-galactosidase and the expression of SIRT1,P53,P21,CDK2,CDK6 and SASP inflammatory factors were tested by above experimental methods.(5)After miR-22 inhibitor was transiently transfected into BNL.CL2 cells,the cells were exposed to PA for 84 hours,β-galactosidase and the expression of SIRT1,P53,P21,CDK2,CDK6 and SASP inflammatory factors were tested by above experimental methods.Results(1)The BALB/c mice fed on high-fat diet showed lipid deposition such as the increase of body weight,liver weight,and epididymis fat weight.Liver triglyceride(TG)level increased;H&E staining showed hepatic fatty degeneration and abnormal lipid metabolism.(2)In HFD group,senescence related β-galactosidase and senescence-associated protein were increased.Immunofluorescence staining showed the expression of P53 was increased,SIRT1 down-regulated.Real-time quantitative PCR showed increased miR-22 expression and decreased Sirt1 expression was detected in HFD group.The sera inflammatory factors IL6 and TNFα were significantly increased in HFD group.(3)β-galactosidase staining showed that the blue-stained positive aging cells were increased in HFD group.Real-time quantitative PCR showed that the expression of Sirt1 and cell cycle genes Cdk2,Cdk6 were down-regulated,P53,P21 up-regulated in BNL.CL2 cells treated with PA.The expression of miR-22 and SASP inflammatory factors were up-regulated.Immunofluorescence staining showed that the expression of P53 and P21 was increased in the nucleus of BNL.CL2 cells treated with PA.The expression of SIRT1 was decreased in the nucleus and increased in the cytoplasm of BNL.CL2 cells.(4)After miR-22 mimics were transiently transfected to BNL.CL2 cells.SIRT1 expression was down-regulated and expression in nucleus was less.Senescence-associated β-galactosidase and senescence-associated genes and proteins were slightly increased.Cell cycle related genes decreased.And SASP inflammatory factor expression increased.(5)After BNL.CL2 cells treated with miR-22 inhibitor and PA,the expression of senescence-associated β-galactosidase was reduced.SIRT1 expression was increased.And mRNA expression of cell cycle related genes was increased.Aging-related genes and proteins were inhibited compared with PA treated group.Conclusion High-fat conditions can induce high expression of miR-22 in hepatocytes.MiR-22 inhibit the activity of SIRT1,leading to SASP up-regulation and induction of hepatocyte senescence.MiR-22 can make the NAFLD progress through targeting SIRT1/SASP,which may become a potential molecular target for the treatment of NAFLD.