GATA4 Prevents Liver Fibrosis And Its Mechanism By Targeting MiR-486-5p

Background:Chronic liver disease caused by different causes can lead to liver fibrosis,which is characterized by a series of events leading to excessive deposition of extracellular matrix proteins such as collagen,scar formation,changes in liver structure and function,leading to organ failure of liver cirrhosis and liver cancer.Although there are significant differences in the pathogenesis and clinical manifestations of fibrotic diseases,the key factor of fiber formation is the activation and proliferation of myofibroblasts,and hepatic stellate cells（HSC）are the main source of myofibroblasts.At present,it is believed that liver fibrosis is a reversible process.Reversing liver fibrosis can effectively delay the progression of patients to cirrhosis and improve the prognosis of patients.A large number of studies have shown that miR-486-5p is a very conservative micro RNA in higher species,which can inhibit myocardial fibrosis,diabetes renal fibrosis,pulmonary fibrosis,lens epithelial cells and trabecular meshwork extracellular matrix synthesis,hypertrophic scar tissue and the development of liver fibrosis.However,the role of miR-486-5p in liver fibrosis and its molecular mechanism have not been fully elucidated.Objectives:This study focused on analyzing the relationship between miR-486-5p and liver fibrosis,and delving into the molecular mechanism of miR-486-5p regulating liver fibrosis.We found that GATA4 targets Smad2 and FBN1 by inducing the expression of miR-486-5p,thereby inhibiting hepatic stellate cell activation and liver fibrosis progression.Therefore,targeting GATA4 and miR-486-5p may represent a potential novel therapeutic approach to halt the progression of liver fibrosis.Our research provides new targets for the clinical treatment of liver fibrosis.Methods:In this project,we first detected the expression of miR-486-5p by real-time fluorescence quantitative PCR（qRT-PCR）in transforming growth factor-β1（TGF-β1）-induced hepatic stellate cell（HSC-T6,LX-2）activation model and carbon tetrachloride（CCl₄）-induced mouse liver fibrosis model.Then,miR-486-5p mimic and inhibitor were used to carry out function acquisition experiment and deletion experiment in hepatic stellate cells.The cell proliferation ability was detected by Ed U staining,the cell cycle was analyzed by flow cytometry,and the fibrosis related marker genes collagen Ⅰ and α-SMA expression were detected by Western blot（WB）,to clarify the effect of miR-486-5p on hepatic stellate cell activation.In vivo,we injected adeno-associated virus 9（AAV9）-mediated miR-486-5p overexpression by the tail vein of mice and induced mouse liver fibrosis model by intraperitoneal injection of CCl₄.The expression of miR-486-5p in liver was detected by qRT-PCR,and the expression of collagen Ⅰ andα-SMA expression to clarify the effect of miR-486-5p on the progression of liver fibrosis.Finally,we predicted the downstream target genes Smad2 and FBN1 of miR-486-5p and the upstream transcription factor GATA4 by bioinformatics method.We carried out function reversal experiment and double luciferase gene reporting experiment in LX-2 cells to clarify the molecular mechanism of miR-486-5p and GATA4 involved in liver fibrosis.Results:1.We found that the expression of miR-486-5p was down regulated in hepatic stellate cell（HSC-T6,lx-2）activation model and mouse liver fibrosis model,suggesting that miR-486-5p may be involved in the development of liver fibrosis.2.The relationship between miR-486-5p and hepatic fibrosis.The results show that:1)in the hepatic stellate cell activation model,miR-486-5p functional reagent（miR-486-5p mimic and inhibitor）interferes with the expression of miR-486-5p.It is found that miR-486-5p overexpression can inhibit cell proliferation and the expression of fibrosis related marker genes,while inhibiting miR-486-5p has the opposite result.2)After successfully constructing the mouse liver fibrosis model in vivo,we found that the overexpression of miR-486-5p in liver can inhibit the deposition of collagen fibers and the expression of fibrosis related marker genes in liver tissue.These data suggest that miR-486-5p can inhibit the development of liver fibrosis.3.The downstream target genes Smad2 and FBN1 of miR-486-5p were predicted by bioinformatics.The results showed that:1)the expression of Smad2 and FBN1 was significantly up-regulated in the hepatic stellate cell activation model,and was negatively regulated by miR-486-5p.2)The results of double luciferase gene report experiment suggest that miR-486-5p has a direct binding site with the 3’UTR region of Smad2 and can inhibit its translation.3)The results of function reversal experiment showed that miR-486-5p inhibited the synthesis and proliferation of extracellular matrix of hepatic stellate cells by targeting the expression of Smad2 and FBN1.4.The upstream regulator GATA4 of miR-486-5p was predicted by bioinformatics.The results showed that:1)GATA4expression was significantly up-regulated in hepatic stellate cell activation model and positively regulated the expression of miR-486-5p.2)The results of functional gain experiment and functional loss experiment showed that overexpression of GATA4could inhibit the activation of hepatic stellate cells,while inhibition of GATA4expression had the opposite result.3)The results of double luciferase gene report experiment suggest that GATA4 has a direct binding site with the promoter region of miR-486-5p and can promote its transcription.4)The results of function reversal experiment showed that GATA4 inhibited the activation of hepatic stellate cells by positively regulating the expression of miR-486-5p.Conclusion:GATA4-induced miR-486-5p inhibits the activation of hepatic stellate cells by suppressing Smad2 and FBN1,thus inhibiting the progression of liver fibrosis.