Depletion of MicroRNA-373 Represses the Replication of Hepatitis C Virus via Activation of Type 1 Interferon Response by Targeting IRF5

Abstract

Purpose: Hepatitis C virus (HCV) poses a risk of chronic liver disease and threatens a significant number of people worldwide. MicroRNAs (miRNAs) are linked to the regulation of hepatocarcinogenesis. Although miR-373 is required for HCV infection, the underlying mechanisms of miR-373 involvement in HCV replication remain elusive.

Materials and methods: Quantitative reverse transcription PCR assays were performed to detect the abundances of miR-373 and HCV RNA either in Huh 7.5 cells or liver biopsy specimens with HCV infection. Luciferase assay was employed to probe the interactions between miR-373 and interferon regulatory factor 5 (IRF5). Western blot was conducted to investigate the effect of miR-373 and IRF5 on HCV replication and activation of type 1 interferon (IFN) response in JFH1-infected Huh 7.5 cells.

Results: HCV infection appeared to be caused by increased miR-373 expression. Addition of miR-373 promoted HCV RNA expression, while miR-373 depletion led to an inhibitive effect on HCV replication. Concordantly, IRF5, as a direct target, was limited by miR-373 in JFH1-infected Huh 7.5 cells. In addition, introduction of IRF5 protected HCV replication in the presence of abundant miR-373. Furthermore, the miR-373-mediated inhibitory effect on type 1 IFN response was ablated following IRF5 accumulation.

Conclusion: miR-373 abrogation reduced HCV replication via activation of type 1 IFN responses by targeting IRF5 in JFH1-infected Huh 7.5 cells, suggesting a promising therapeutic for treating HCV infection.

Keywords: Hepatitis C virus; IRF5; miR-373; replication; type 1 IFN.

Fig. 1. miR-373 up-regulated in liver tissues and Huh 7.5 cells with HCV infection. (A) The expression of miR-373 was detected in primary human hepatocytes treated by mock or in HCV-infected cells for 96 h. (B) The expression of miR-373 was detected in Huh 7.5 cells treated by mock or in JFH1-infected cells for 96 h. (C) The expression of miR-373 was detected in liver biopsy specimens from patients with or without HCV infection. (D) Correlation plot for HCV RNA expression versus miR-373 levels in liver biopsy specimens with or without HCV infection. Data represent the mean±standard deviation from at least three independent experiments. ^*p<0.05. HCV, hepatitis C virus.

Fig. 2. Overexpression of miR-373 promotes HCV replication in JFH1-infected Huh 7.5 cells. (A and B) The expression of miR-373 and HCV RNA were investigated in JFH1-infected Huh 7.5 cells transfected with miR-373 mimics or inhibitor by quantitative reverse transcription PCR. (C and D) The expressions of NS3 and NS5A were analyzed in JFH1-infected Huh 7.5 cells transfected with miR-373 mimics or inhibitor by Western blot. Data represent the mean±standard deviation from at least three independent experiments. ^*p<0.05. HCV, hepatitis C virus.

Fig. 3. IRF5 is a target of miR-373. (A) Wild-type (IRF5-wt-3′-UTR) and mutant (IRF5-mt-3′-UTR) of putative miR-373 targeting sequences. (B) Analysis of luciferase activity was performed in 293T cells transfected with miR-373 mimics and IRF5-wt-3′-UTR or IRF5-mt-3′-UTR, compared with negative control. (C) The expression of IRF5 protein was analyzed in JFH1-infected Huh 7.5 cells transfected with miR-373 mimics or inhibitor, compared with NC, by Western blot. Data represent the mean±standard deviation from at least three independent experiments. ^*p<0.05. IRF5, interferon regulatory factor 5; HCV, hepatitis C virus.

Fig. 4. IRF5 attenuates the effect of miR-373 on HCV replication in JFH1-infected Huh 7.5 cells. (A) The effect of IRF5 on miR-373-mediated HCV RNA expression was investigated in Huh 7.5 cells transfected with miR-373, miR-373+IRF5, miR-373+pcDNA, or miR-NC. (B) The effect of IRF5 inhibition on anti-miR-373-regulated HCV RNA expression was measured in Huh 7.5 cells transfected with anti-miR-373, anti-miR-373+si-IRF5, anti-miR-373+si-NC, or anti-miR-NC. (C) The effect of IRF5 on miR-373-mediated protein expression of NS3 and NS5A was investigated in Huh 7.5 cells transfected with miR-373, miR-373+IRF5, miR-373+pcDNA, or miR-NC. (D) The effect of IRF5 interference on anti-miR-373-mediated regulation of NS3 and NS5A protein levels was evaluated in Huh 7.5 cells transfected with anti-miR-373, anti-miR-373+si-IRF5, anti-miR-373+si-NC, or anti-miR-NC. Data represent the mean±standard deviation from at least three independent experiments. ^*p<0.05. IRF5, interferon regulatory factor 5; HCV, hepatitis C virus.

Fig. 5. miR-373-mediated suppression of type 1 IFN response is ablated following addition of IRF5. (A) The expressions of proteins related with type 1 IFN response (PKR, OAS, and MxA) were detected in JFH1-infected Huh 7.5 cells transfected with miR-373 mimics and IRF5. (B) The expression levels of the proteins were detected in JFH1-infected Huh 7.5 cells transfected with miR-373 inhibitor and si-IRF5. Data represent the mean±standard deviation from at least three independent experiments. ^*p<0.05. IFN, interferon; MxA, myxovirus protein A; OAS, 2′–5′-oligoadenylate synthetases; PKR, double-stranded RNA-dependent protein kinase; IRF5, interferon regulatory factor 5.

Fig. 6. Schematic figure shows the relationship between HCV and miR-373. HCV infection induces miR-373 abundance in HCV tissues and JFH1-infected Huh 7.5 cells. Moreover, miR-373 inhibits IRF5 expression, which in turn activates the type I IFN response to limit HCV replication. IRF5, interferon regulatory factor 5; HCV, hepatitis C virus.