miR-29a Promotes Lipid Droplet and Triglyceride Formation in HCV Infection by Inducing Expression of SREBP-1c and CAV1.

Aims: To examine the regulation of SREBP-1c and CAV1 by microRNA-29a (miR-29a) in cells infected with hepatitis C virus (HCV) in an attempt to control HCV-induced non-alcoholic fatty liver disease. Methods: In order to examine the manipulation of SREBP-1c and CAV1 by miR-29a, oleic acid (OA)-treated JFH-I-infected Huh-7 cells were used. OA was added 24 h post-transfection and gene expression was investigated by qRT-PCR at 48 h post treatment. The functional impact of the observed alteration in SREBP-1c and CAV1 expression was analyzed by examining lipid droplet (LD) and triglyceride (TG) content at 72 h post-OA treatment using light microscopy and spectrophotometry, respectively. Viral load was quantified by qRT-PCR at 72 h post-transfection. Results: OA treatment induced the expression of miR-29a and SREBP-1c, as compared to untreated cells. Forced miR-29a expression led to a significant up-regulation of SREBP-1c as well as CAV1 compared to mock untransfected cells. Ectopic expression of miR-29a resulted in a marked increase in LDs and their respective TGs, while miR-29a antagomirs decreased both the LD and TG content compared to mock untransfected cells. Moreover, forcing the expression of miR-29a in JFH-1 HCV-infected Huh-7 cells resulted in 53% reduction in viral titers compared to mock untransfected Huh-7 cells. Conclusion: Inducing miR-29a expression significantly induces SREBP-1c and CAV1 expression, thereby increasing LDs as well as their respective TGs. Nonetheless, forcing the expression of miR-29a resulted in reduction of HCV RNA levels in Huh-7 cells.

Repressing PU.1 by miR-29a∗ in NK cells of HCV patients, diminishes its cytolytic effect on HCV infected cell models.

OBJECTIVES: Natural killer cells are immune safeguards against HCV infection. PU.1 is a pivotal transcription factor in the development of NK cells. This study aimed at studying the regulatory effect of miRNAs on both development and function of NK cells isolated from HCV patients. METHODS: NK cells were isolated from 17 chronic HCV patients and 12 healthy controls; after which miRNA and mRNA were quantified using qRT-PCR. Manipulating miRNA expression using mimics and antagomirs, was performed followed by investigating downstream targets as well as viral abundance. RESULTS: PU.1 expression levels were upregulated in NK cells of HCV patients. In silico analysis revealed PU.1 to be a potential downstream target of miR-29a(∗), where miR-29a(∗) overexpression in NK cells caused a significant downregulation in PU.1 mRNA. Forcing miR-29a(∗) caused a downregulation of the cytotoxicity determinant NK activating receptor (NKG2D) via upregulation of miR-155. Moreover, perforin-1 mRNA was found to be downregulated upon forcing the expression of miR-29a(∗) in NK cells of HCV patients. This decrease in NK cytolytic function was accompanied by an 80% viral load increase in cocultured HCVcc cell models. CONCLUSIONS: This study showed that HCV infection might abrogate NK cytotoxic potential through altering PU.1, NKG2D receptor and perforin molecules.

Mir-194 is a hepatocyte gate keeper hindering HCV entry through targeting CD81 receptor.

OBJECTIVE: The tetraspanin CD81 is one of the main receptors involved in hepatitis C virus entry. Herein, we aimed to explore the role of microRNAs in regulating CD81 receptor expression and function. PATIENTS AND METHODS: Bioinformatics analysis was carried out to select potential mircroRNAs that binds CD81 3'untranslated region. Liver biopsies taken from 28 HCV genotype- 4 patients and 10 healthy donors were screened. Naïve, JFH1 and ED43/JFH1- infected- Huh7 cells were transfected with mimics and inhibitors followed by analyzing CD81 protein and mRNA expression. This was done using flow cytometry and Q-RT PCR, respectively. HCV entry into Huh7 cells was investigated post-transfection. Binding confirmation was done using luciferase reporter vector harboring wild/mutant target sites of microRNA. The impact of Epigallocatechin-gallate on modulating microRNA/CD81 expression was assessed. RESULTS: Bioinformatics revealed that CD81 is a potential down-stream target for miR-194. A significant inverse correlation was found between miR-194 and CD81 expression in liver biopsies of HCV patients. Forcing the expression of miR-194 showed a down-regulation of CD81 protein, mRNA expression and significantly abrogated the HCV infectivity of Huh7 cells. Stimulation with EGCG enhanced mir-194 expression and down-regulated CD81 expression. CONCLUSION: This study showed that mir-194 hinders HCV entry through targeting CD81 receptors.