Differential Expression And Mechanism Of MiRNA In Hepatic Fibrosis Induced By Biliary Obstruction

Objective:Liver fibrosis is a chronic injury and repair response in the liver caused by various factors.Persistent liver fibrosis could develop into hepatic cirrhosis and even cancer,gradually.Micro RNAs(miRNAs)are a class of non-coding RNAs consisting of 18-24 nucleotide molecules.Study found that miRNA may be involved in the occurrence and development of liver diseases.We aim to explore the differential expression of miRNAs in obstructive hepatic fibrosis and the biological function and specific mechanism of miR-183-5p in hepatic fibrosis.Methods:Collection of 3 cases of liver fibrosis induced by intrahepatic bile duct stones and 3cases of normal liver tissue samples,and the differentially expressed miRNAs in liver tissues were detected and identified by high-throughput sequencing.miR-183-5p expression levels in rescreened liver fibrosis and normal liver tissue samples were detected by RT-q PCR.Forty male Wistar rats were selected and randomly divided into 4 groups.We separated the common bile duct from the surrounding tissues without ligation in the shamoperated group.The rats in the BDL group were modeled by ligating the common bile duct for 2 weeks.The miR-183-5p antagomir group was injected with miR-183-5p antagomir through the tail vein,and the control antagomir group was injected with the control reagent in the same way.Rats were sacrificed 2 weeks after operation to obtain liver and serum samples.RT-q PCR was used to detect the level of miR-183-5p in liver,the liver pathological changes and collagen deposition in liver were detected by HE and Masson staining.The levels of total bilirubin(TBIL),aspartate aminotransferase(AST)and alanine aminotransferase(ALT)in serum were measured using an automatic biochemical analyzer.Western blotting was used to detect α-SMA,collagen-I and TIMP-1 expression in the liver.Immunohistochemistry was used to detect α-SMA distribution in liver tissue.LX-2 cells were divided into 4 groups,LX-2 cells were transfected with miR-183-5p antagomir and control antagomir reagents using Lipofectamine 2000.RT-q PCR was used to detect the levels of miR-183-5p and α-SMA in LX-2 cells,and the expression of α-SMA,collagen-I,and TIMP-1 in cells of each group were detected by western blot,The cell viability of LX-2 was assessed with CCK-8 kit,and the apoptosis of cells were detected by flow cytometry.The target genes of miR-183-5p were predicted by miRbase,miRDB and Target Scan,the expression levels of FOXO1 m RNA in humans,rats and cells were detected by RT-q PCR,and the relationship between miR-183-5p and FOXO1 was analyzed by Pearson correlation.The distribution of FOXO1 in rat liver tissue was determined by histochemical method.Next,the interaction between miR-183-5p and FOXO1 was further identified by dual luciferase assay.We then divided TGF-β1-activated LX-2 cells into 4 groups.We transfected miR-183-5p agomir,control agomir reagent and FOXO1 lentivirus(LV-FOXO1)into cells using Lipofectamine 2000.We used RT-q PCR to detect miR-183-5p and α-SMA expression.Western blot was used to detect α-SMA,collagen-I,TIMP-1,FOXO1,TGF-β,p-smad2/3,Smad2 and Smad3 expression in each group,the cell viability of LX-2 in each group was evaluated by CCK-8 kit,and the apoptosis of cells were detected by flow cytometry.Results:In our study,we identified a total of 44 miRNAs with statistically significant differences by high-throughput sequencing,including 18 up-regulated and 26 downregulated miRNAs.The expression of miR-183-5p was increased in the re-screened human fibrotic liver tissue samples detected by RT-q PCR,which matched the previous sequencing results.In addition,RT-q PCR results showed that miR-183-5p was upregulated in both rat liver fibrosis model and activated LX-2 cells.The application of miR-183-5p antagomir reduced the level of miR-183-5p,attenuated liver fibrosis,down-regulated the α-SMA,collagen-I,and TIMP-1 expression in vitro and in vivo,and inhibited LX-2 cells proliferated and induced apoptosis.The dual-luciferase assay showed that miR-183-5p inhibited the expression of FOXO1 by directly binding to its 3’-UTR.Next,we found that LV-FOXO1 could overexpress FOXO1 in LX-2 cells,and that overexpression of FOXO1 reversed the promoting effect of miR-183-5p on liver fibrosis and downregulated the protein levels of SMA,collagen-I and TIMP-1 in LX-2 cells in each group,and inhibited cell proliferation and promoted apoptosis.Western blot results showed that overexpression of FOXO1 inhibited the expression of TGF-β signaling pathway proteins and inhibited the activation of this pathway.Conclusion:Differentially expressed miRNAs exist in fibrotic livers caused by biliary obstruction,and miR-183-5p may be a key factor regulating liver fibrosis.miR-183-5p promotes cholestatic liver fibrosis by inhibiting FOXO1 expression and activating the TGF-βsignaling pathway.In summary,inhibiting miR-183-5p may be a new approach to prevent and improve liver fibrosis.