MicroRNA-125a Impaired Autophagic Flux By Targeting The Vitamin D Receptor In Hepatocytes And May Contribute To Fibrosis In A CCL4-induced Mouse Model And Patients With Liver Fibrosis

Background and Aims Hepatic fibrosis refers to the pathological process of abnormal proliferation of fibrous connective tissue in the liver during the necrosis or inflammatory stimulation of hepatocytes,which is the common pathological basis of all chronic liver diseases.There is abundant evidence that autophagy has a protective effect on the anti-fibrosis process of the liver.In recent years,miRNAs and vitamin D receptors(VDR)have been proved to be highly involved in the regulation of liver fibrosis and autophagy.This study intends to elucidate the role and molecular mechanism of miR-125 a /VDR axis in the regulation of autophagic flux,hepatocyte injury and liver fibrosis.Methods 60 liver biopsy samples from patients with liver fibrosis were collected,and inclusion criteria were as follows: pathological diagnosis of liver fibrosis,etiology included HBV/HCV infection,alcoholic fibrosis,fatty liver fibrosis,and autoimmune fibrosis.5 normal liver tissue samples were used as negative control.miR-125a-antgomir-GFP AAV and VDR sh RNA AAV were applied to down-regulate miR-125 a and VDR respectively by tail vein injection.HE staining,Masson staining,immunohistochemistry,real-time quantitative PCR,Western blot and luciferase reporter gene analysis were applied to evaluate the effects of miR-125 a /VDR axis on autophagic flux,hepatocyte injury and liver fibrosis.Results Compared with the control group,the expression of VDR in hepatocytes of liver tissue from liver fibrosis patients and CCL4-induced liver fibrosis mice model was down-regulated,accompanied by impaired autophagic flux(increased LC3 expression and P62 accumulation).The degree of liver fibrosis was negatively correlated with the expression of VDR and the autophagic flux in hepatocytes.The results of GEO microarray database indicated that the expression level of miR-125 a in human liver tissue samples form patients with alcoholic fibrosis,viral hepatitis fibrosis,or CCL4-induced hepatic fibrosis mouse model was higher than that in the normal control group.Luciferase analysis confirmed that VDR was a direct target of miR-125 a.The level of miR-125 a in human fibrosis tissues was 3.23 times higher than that in normal people,and was negatively correlated with autophagic flux and VDR expression.Down-regulation of miR-125 a expression with antagomir-GFP AAV partially restored VDR expression and improved autophagic flux in hepatocytes.In addition,antagomir-GFP AAV alleviated hepatic fibrosis and α-SMA expression in CCL4-induced hepatic fibrosis mouse model.Knockdown VDR expression with VDR sh RNA AAV could exert the antagonistic effect of miR-125a-antagomir-GFP AAV on the impairment of autophagic flux and fibrosis in CCL4-induced mouse model.Conclusions In liver fibrosis patients and CCL4-induced liver fibrosis mouse models,miR-125 a negatively regulates the expression of vitamin D receptor in hepatocytes,leading to impaired autophagic flux and hepatocyte injury,and ultimately leading to the progression of hepatic fibrosis.