The Mechanism Of Huh7Migration And HCV Replication Affected By MiR-141

Objective：microRNAs (miRNA) are a group of small, noncoding single-stranded RNAmolecules(18-22nt in length) that encoded by endogenous gene and regulate the expressionof target genes by interfering with transcription or inhibiting translation. Studies havedemonstrated that miRNAs play a crucial role in almost all cellular biological processincluding metabolism, survival differentiation and apoptosis. Deregulation of specificmiRNAs contributes to a variety of diseases, most remarkly in cancer development andprogression. miRNA expression profiling in hepatocellular carcinoma(HCC) showed that anumber of miRNAs，including miR-141were downregulated in tumor tissues, but little isknown about the function of miR-141in HCC migration. The purpose of this study is toexplore whether miR-141played an important role in HCC migration or not. Furthermore,since HCV chronic infection associates with HCC development, how miR-141regulatingHCV replication remains elusive, we try to identify the link between miR-141and HCVreplication and whether IFN-β signaling pathway is involved.Methods:1. Using Lentiviral vectors to establish Huh7-miR-141-LV, which stably overexpressmiR-141in Huh7, and Huh7-NC-LV as a negative control.2. Using transwell assay to explore whether miR-141played an important role in HCCmigration or not.3. qRT-PCR was used to determine the titer of JFH1in cell culture supernatant after JFH1infected Huh7with a certain time course.4. qRT-PCR and Western Blot were used to determine the mRNA and protein expressionlevel of related genes in TGF-β and TLR/IRF signaling pathways.5. Dual-Luciferase reporter gene assay was used to determine IFN-β Luciferase activityafter JFH1infection.Results：1. Successfully established Huh7-miR-141-LV, which could stably overexpress miR-141inHuh7.2. miR-141overexpressed in Huh7could inhibit the ability of Huh7migration and whenmiR-141was inhibited, the ability of Huh7migration was comeback. 3. miR-141could block TGF-β signaling pathway by down-regulating TGF-β R1expression, which is a key gene in TGF-β signaling pathway. Overexpression of miR-141inHuh7also inhibit the expression of MMP2and MMP9.4. When miR-141was overexpressed in Huh7, the ability of HCV strain JFH1replication inHuh7was inhibited.5. After JFH1infect Huh7-miR-141-LV, mRNA expression level of TLR7, TLR9and IRF7and the protein expression level of IRF3are highly promoted by overexpression of miR-141.6. Dual-Luciferase reporter gene assay found that IFN-β Luciferase activitity was higher inHuh7-miR-141-LV than Huh7-NC-LV not only before but also after JFH1infection.Conclusion:1. Our study showed that miR-141could inhibit Huh7migration by inhibiting the expressionof TGF-β R1, which is a key gene in TGF-β signaling pathway. When miR-141wasoverexpressed in Huh7, both mRNA and protein expression level of MMP2and MMP9wereinhibited. The next step of our research is to make it clear that the link between miR-141andMMP2, MMP9in HCC migration.2. JFH1infection could active TLR/IRF signaling pathway and promote the transcription ofIFN-β gene, thus establish antiviral status. Our data show that overexpressing miR-141could inhibit the ability of JFH1replication in Huh7, and this inhibition might be regulatedby increasing TLR7, TLR9, IRF3, IRF7expression level, and activating IFN-β, but themechanism of how miR-141promoting these related genes expression is not clear, so weneed to do more researches.