Mutation Of MiR-21 Targets Endogenous Lipoprotein Receptor-related Protein 6 In The Pathogenesis Of Nonalcoholic Fatty Liver Diseaseand Nonalcoholic Fatty Liver Disease

Objective:Nonalcoholic fatty liver disease(NAFLD)is a chronic liver disease characterized by liver fat accumulation and abnormal lipid metabolism.With the global transformation of lifestyle,the population of NAFLD increased rapidly,has become one of the most important public health problems in the 21 st century,which has brought great economic burden to society.In recent years,a large number of studies have revealed that micro RNA(mi RNA)spectrum changes in NAFLD,to explore the emerging molecular mechanism of miRNA target treatment of NAFLD and related disorders has attracted the attention of researchers.It has been reported that mi R-21 and low-density lipoprotein(LDL)receptor-related protein 6(LRP6)are involved in the pathogenesis of NAFLD,but whether mi R-21 can be used as a therapeutic target or the relationship between miR-21 and LRP6 are unknown.In this study,in vitro experiments were performed using a cell model of NAFLD to study the effect of miR-21 on lipid synthesis and secretion,to determine the the new target of miR-21 in the roles of NAFLD.Methods:(1)HepG2 cells were intervened by palmitic acid(PA)and oleic acid(Oleic acid,OA)(1: 2,v / v)mixture(PA/OA)to establish the vitro cell model of NAFLD,miR-21 expression in the PA/OA intervened HepG2 cells was silenced by siRNA technique,and miR-21 expression was up-regulated with miR-21 mimic or control mimic transfected,then the effects of mi R-21 on the synthesis and secretion of cholesterol(C),cholesteryl ester(CE),triglyceride(TG)and phospholipid(PL)were monitored;(2)miR-21 mimic or control mimic,miR-21 antagomir or control antagomir transfected the PA/OA intervened Hep G2 cells up-regulated and blocked miR-21 expression,then monitored the expression of LRP6,respectively;(3)Quantitative real-time PCR and Western blot analysis were used to detect the genes involved in lipid metabolism,including liver X receptor ?(LXR?),stearoyl-CoA desaturase 1(SCD1),Acetyl-CoA carboxylase1(ACC1)and Sterol regulatory element binding protein 1(SREBP1),as well as the expression of LRP6 at mRNA and protein levels;(4)Run Target Scan programs,then the LRP6 3'untranslated region(UTR)or LRP6 mutant 3'-UTR or LXRa 3'-UTR as a negative control was subcloned into a luciferase reporter vector,respectively,with miR-21 mimic or control mimic co-transfected the PA/OA intervened Hep G2 cells.Dual luciferase reporter assays were conducted to confirm the effects of miR-21 expression levels on LRP6.Results:(1)miR-21 mimic transfected the PA/OA intervened Hep G2 cells increased the levels of cholesterol,triglyceride,and phospholipid,but not cholesteryl ester.Quantitative real-time PCR showed that miR-21 up-regulation could induce the expression of genes encoding lipogenic enzymes,including ACC1,SCD1,SREBP1,and LXR?,but not fatty acid synthase(FASN);(2)mi R-21 mimictransfected the PA/OA intervened HepG2 cells,quantitative real-time PCR and western blot showed LRP6 expression in mRNA and protein levels decreased.In contrast,miR-21 antagomir transfection increased the expression level of LRP6;(3)Run Target Scan programs showed that LRP6 was a potential target for miR-21.Dual luciferase reporter assay showed that luciferase activity was significantly reduced in LRP6 3'-UTR and miR-21 mimic co-transfected the PA/OA intervened HepG2 cells,but transfection of miR-21 mimic had no effect on luciferase activity of LRP6 mutant 3'-UTR and LXRa 3'-UTR.Conclusion:miR-21 regulates lipid metabolism in vitro cell model of NAFLD,which is achieved by inhibiting the expression of LRP6.LRP6 is a new target for miR-21.miR-21 may be a new strategy for treating NAFLD by targeting endogenous LRP6.