Hepatitis C virus infection modulates expression of interferon stimulatory gene IFITM1 by upregulating miR-130A - PubMed (original) (raw)

Hepatitis C virus infection modulates expression of interferon stimulatory gene IFITM1 by upregulating miR-130A

Joydip Bhanja Chowdhury et al. J Virol. 2012 Sep.

Abstract

We have examined the underlying mechanism of hepatitis C virus (HCV)-mediated IFITM1 regulation. IFITM1 is a potential target of miR-130a. Our results demonstrated that miR-130a expression was significantly higher in HCV-infected hepatocytes and liver biopsy specimens than in controls. Introduction of anti-miR-130a in hepatocytes increased IFITM1 expression. Hepatocytes stably expressing IFITM1 reduced HCV replication. Together, these results suggested that HCV infection of hepatocytes upregulates miR-130a and that use of anti-miR-130a may have potential for restriction of HCV replication.

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Figures

Fig 1

HCV infection enhances miR-130a expression. (A) Immortalized human hepatocytes (IHH) were infected with HCV genotype 1a for 72 h. RNA was isolated, and miR-130a was measured by real-time PCR. U6 RNA was used as an internal control. The results from three independent experiments are presented. (B) Total RNA was extracted from HCV-infected and control liver biopsy specimens (not infected with HCV or HBV). miR-130a expression was analyzed by real-time PCR, and U6 RNA was used as an internal control. A comparison of miR-130a levels in control (n = 8) and HCV-infected (n = 9) liver tissues is shown. (C) Results from individual specimens of panel B are shown. HCV loads from each specimen are mentioned on the top of each bar. (D) IFITM1 expression from control (n = 6) and HCV-infected (n = 6) liver biopsy specimens after normalization with internal control 18S RNA is shown.

Fig 2

Knockdown of miR-130a enhances IFITM1 expression in hepatocytes. (A) Huh7 cells were treated with a control or anti-miR-130a. Cell lysates were prepared after 48 h of transfection, and IFITM1 expression was analyzed by a Western blot using a specific antibody. The blots were reprobed with an antibody to actin for a comparison of protein loads. The fold change of IFITM1 after normalization with actin is shown and compared with the control-treated sample. (B) IFITM1 mRNA was measured from control and miR-130a knockdown cells as described in Fig. 1. The results are the means of three independent experiments. (C and D) Huh7.5 cells were infected with HCV and were treated with the control or anti-miR-130a. HCV and IFITM1 mRNA levels were measured by real-time PCR and presented after normalization with GAPDH. The results shown are from two independent experiments with three replicates. (E) HCV-infected cells transfected with the control or anti-miR-130a. Cells were examined for miR-130a expression by qRT-PCR. Results after normalization with U6 as an internal control are presented.

Fig 3

miR-130a targets IFITM1 in hepatocytes. (A) Computational analysis of the IFITM1 3′ UTR revealed a single putative miR-130a binding site. The miR-130a target region of IFITM1 (GenBank accession number NM_003641.3) is indicated. (B) Luciferase assay showing decreased reporter activity after cotransfection of the IFITM1 3′ UTR and mimic miR-130a. On the other hand, the mutant 3′ UTR of IFITM1 had no detectable effect on reporter activity. The results are the means ± the standard errors from three independent experiments.

Fig 4

Exogenous expression of IFITM1 rescues the miR-130a effect. Huh7 cells were stably transfected with IFITM1 or vector control, followed by HCV genotype 2a infection. Cells were incubated for 72 h. (A) Total RNA from cells was isolated and analyzed for HCV replication after normalization with GAPDH. (B) miR-130a expression status was examined from total RNA by real-time PCR as described in Fig. 1. The results from two independent experiments with three replicates are shown.

Fig 5

Schematic diagram showing the cross talk among HCV, miR-130a, and IFITM1. Enhanced IFITM1 expression inhibits HCV replication. HCV infection upregulates miR-130a in hepatocytes, which, in turn, inhibits IFITM1 expression. On the other hand, HCV infection inhibits alpha interferon (IFN-α)-induced IFITM1 expression. Arrows denote upregulation, and blunt arrows denote inhibition.

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Hepatitis C virus infection modulates expression of interferon stimulatory gene IFITM1 by upregulating miR-130A - PubMed (original) (raw)