Alterations in microRNA expression profile in HCV-infected hepatoma cells: Involvement of miR-491 in regulation of HCV replication via the PI3 kinase/Akt pathway

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Abstract

The aim of this study was to investigate the role of microRNA (miRNA) on hepatitis C virus (HCV) replication in hepatoma cells. Using miRNA array analysis, miR-192/miR-215, miR-194, miR-320, and miR-491 were identified as miRNAs whose expression levels were altered by HCV infection. Among them, miR-192/miR-215 and miR-491 were capable of enhancing replication of the HCV replicon as well as HCV itself. HCV IRES activity or cell proliferation was not increased by forced expression of miR-192/miR-215 or miR-491. Investigation of signaling pathways revealed that miR-491 specifically suppressed the phosphoinositol-3 (PI3) kinase/Akt pathway. Under inhibition of PI3 kinase by LY294002, the suppressive effect of miR-491 on HCV replication was abolished, indicating that suppression of HCV replication by miR-491 was dependent on the PI3 kinase/Akt pathway. miRNAs altered by HCV infection would then affect HCV replication, which implies a complicated mechanism for regulating HCV replication. HCV-induced miRNA may be involved in changes in cellular properties including hepatocarcinogenesis.

Highlights

HCV infection upregulated miR-192, -194, -215, downregulated miR-320, -491. ► Transfection of miR-192, -215, and -491 enhanced HCV replication. ► Transfection of miR-491 inhibited Akt phosphorylation. ► Akt inhibition could be responsible for augmentation of HCV replication by miR-491.

Introduction

Hepatitis C virus (HCV) is a major causative agent of liver diseases worldwide. Elimination of HCV fails in about 80% of infected patients, which leads to chronic hepatitis, liver cirrhosis, and subsequent development of hepatocellular carcinoma [1]. Combination therapy of pegylated-interferon-α and ribavirin results in sustained clearance of serum HCV-RNA in only ∼50% of patients [2], [3]. To improve therapeutic efficacy of the virologic response rate, drugs inhibiting the functions of HCV proteins such as NS3, NS5A, and NS5B, are currently under development. Although a number of studies have clarified the mechanisms of the effect of HCV on infected cells or the role of host factors on regulation of HCV replication, there remains much to be investigated.

MicroRNAs (miRNAs) were identified as a population of small RNAs, modulating translation by binding to sites of antisense complementarity in 3′ untranslated regions of target mRNA [4]. With respect to regulation of HCV replication, the relevance of several miRNAs has been recently reported. miR-122, a hepatocyte-specific miRNA, was identified as a positive regulatory factor for HCV replication by binding to two sites in the HCV genome [5]. Each of the interferon-β-induced miRNAs, miR-196, miR-296, miR-351, miR-431, and miR-448, has a partially complementary sequence to HCV, resulting in suppression of HCV replication [6]. Thus, a miRNA with homology to the HCV sequence is likely to have the ability to regulate HCV. Another possible mechanism of miRNA regulation of HCV replication is the targeting of some cellular gene involved in HCV replication. miR-141 was shown to suppress DLC-1 leading to efficient HCV replication [7]. Although some miRNAs were shown to be capable of regulating HCV replication, details of the relationship between miRNAs and HCV replication are still largely unknown.

In the present study, we performed miRNA array analysis to identify miRNA(s) altered by HCV infection in Huh7, a hepatoma cell line. We further investigated whether HCV-regulated miRNA could, in turn, affect HCV replication. As a result, we were able to identify five miRNAs: miR-192 and its homolog miR-215 and miR-194 as upregulated miRNAs and miR-320 and miR-491 as downregulated miRNAs. Among them, miR192/miR-215 and miR-491 enhanced HCV replication in HCV replicon cells as well as in cell culture-infectious HCV (HCVcc)-infected cells. miR-192/miR215 and miR-491 did not increase cell proliferation or HCV internal ribosome entry site (IRES) activity, suggesting that these were not the reasons for increased HCV replication. Further investigation revealed that miR-491 suppressed the PI3 kinase/Akt pathway suggesting that this could be responsible for augmentation of HCV replication by miR-491.

Section snippets

Cells, antibodies

The hepatoma-derived cell line Huh7 was maintained in DMEM supplemented with 10% FCS. The HCV subgenomic cell line Huh-RepSI, harboring HCV-N (genotype 1b), was previously described [8]. Antibodies to phospho-ERK (Thr202/Tyr204), Akt, phospho-Akt (Ser473) were purchased from Cell Signaling Technology. An antibody to β-actin (A-5441) was from Sigma–Aldrich. A mouse monoclonal antibody to HCV core protein (C7-50) was obtained from Affinity BioReagents. A mouse monoclonal antibody to HCV NS5A

Identification of miRNAs regulated by HCV infection

Huh7 cells were infected with HCVcc at ∼2 m.o.i. After incubation for 5 days, total RNA was extracted from the cells followed by purification with small RNA and miRNA array analysis. A portion of the cells was subjected to immunofluorescence analysis for staining of HCV core protein to verify that more than 90% of the cells were infected with HCV. The ratio of Cy3 intensity to Cy5 intensity was calculated and alteration of the miRNA expression profile was analyzed. A ratio of more than 1.5-fold

Discussion

In the present study, we tried to identify the miRNA(s) affected by HCV infection and establish how they influence HCV replication. Five miRNAs, miR-192, miR-194, miR-215, miR-320, and miR-491, were identified as HCV-regulated miRNAs by miRNA array analysis. Three upregulated miRNAs, miR-192, miR-194, and miR-215, were previously identified as p53-inducible miRNAs [12], [13]. Two miRNA clusters which encode identical miR-194 sequences (i.e., the miR-194-2/miR-192 cluster on chromosome 1) and

Acknowledgment

We thank Stanley Lemon for providing the plasmid pRLHL and the cell culture-infectious virus HJ3-5(YH/QL).

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