The Mechanism Of MiR-141 Regulating HCV Infection In Hepatocellular Carcinoma Cells

AIM: To investigate the role of micro RNA-141 in the process of HCV infection,mainly from two aspects: the regulation mechanism of micro RNA-141 on the entry of HCV into host cells,the change of IFN ? signaling pathway and the effect of HCV replication.METHOD: Quantitative RT-PCR was used to detect the expression of micro RNA-141 and related genes;Western blot and immunohistochemistry were used to detect the expression of Eph A2 and related genes;HCV particle detection kit was used to determine the viral titer of prepared viruses and the viral particles released from the supernatant of treated cells;HCV particles entering cells and cell surface were detected by immunofluorescence.Target Scan,Pic Tar,Star Base and other online software were used to predict the target gene of micro RNA-141,UCSC and Reg RNA 2.0 were used to predict the promoter sequence of the target gene and find the binding sites of the target gene to micro RNA-141,and double luciferase reporter gene test was used to verify the targeting relationship between micro RNA-141 and Eph A2 and the activity of IFN ? and NF-?B p65.Eph A2 and TLR8 were knocked down by si RNA to analyze the similarity of biological effects between knockdown of Eph A2 and overexpression of micro RNA-141;hepatocellular carcinoma cells were treated with dasatinib,a small molecular inhibitor of Eph A2;and the effects of overexpression of micro RNA-141 on HCV transmission mediated by cell-cell contact were detected by co-culture.RESULTS: On the one hand,the expression of miR-141 in HCV-infected positive peripheral blood was very low,and the expression in HCV-infected hepatocellular carcinoma was significantly lower than that in adjacent non-cancer tissues.Overexpression of micro RNA-141 significantly reduced the expression of HCV NS5 A and Core.Immunofluorescence results showed that the expression of HCV Core in the cells overexpressing micro RNA-141 was significantly lower than that in the control cells in the early stage of viral infection.Luciferase reporter assay confirmed that overexpression of micro RNA-141 could significantly inhibit the expression of Eph A2;Eph A2 in the cells overexpressing micro RNA-141 was significantly inhibited.The expression of Eph A2 in tumor tissues caused by infection of HCV was significantly higher than that in adjacent non-cancer tissues;knockdown of Eph A2 by si RNA significantly reduced the expression of HCV NS5 A and Core.After treatment with Dasatinib,a specific inhibitor of Eph A2,the expression of Eph A2 was significantly inhibited,the expression of HCV NS5 A and Core in cells significantly decreased,and the expression of HCV Core into cells decreased.Overexpression of micro RNA-141 inhibited co-localization of Eph A2-CLDN1,Eph A2-OCLN and CD81-CLDN,and overexpression of micro RNA-141 significantly reduced the transmission of cell-to-cell virus.On the other hand,with the long time of HCV infection,the expression of micro RNA-141 gradually decreased;overexpression of micro RNA-141 significantly decreased the expression of HCV Core and NS5A;overexpression of micro RNA-141 significantly increased the expression of IFN ? after HCV infection;overexpression of micro RNA-141 increased the expression of IRF3,c-Jun and NF-?B p65 in IFN ? signaling pathway;Western blot results showed that the expression of IRF3,c-Jun and NF-?B p65 was significantly increased.The expression of IRF3,c-Jun and NF-?B p65 in IFN ? signaling pathway decreased after knockdown of miR-141,while the expression of HCV NS5 A and Core increased.Western blot analysis of non-denaturing protein showed that the formation of IRF3 dimer in cells overexpressing micro RNA-141 increased significantly after HCV infection,while the formation of IRF3 dimer was significantly inhibited after knockdown of micro RNA-141.Overexpression of micro RNA-141 was significantly inhibited in vitro.The expression of TLR7,TLR8 and TLR9 was increased;the software predicted that micro RNA-141 could bind to the promoter region of TLR8 gene,which was mediated by IFN ? signaling pathway;knockdown of TLR8 resulted in a marked increase in the expression of HCV NS5 A and Core,and a decrease in the expression of IRF3,c-Jun and NF-?B p65 and the dimerization of IRF3.CONCLUSION: MiR-141 can reduce the binding of Eph A2 to CLDN1 and OCLN by down-regulating Eph A2,inhibit co-localization of CD81 and CLDN1,reduce the internalization of virus-cell receptor complex,and thus inhibit the entry of HCV into host cells.MiR-141 can further activate the expression of IFN ? signaling pathway by enhancing the expression of TLR8 and then enhance the effect of anti-HCV infection.