miR-27b-mediated suppression of aquaporin-11 expression in hepatocytes reduces HCV genomic RNA levels but not viral titers

Abstract

Background: MicroRNAs (miRNAs) have gained much attention as cellular factors regulating hepatitis C virus (HCV) infection. miR-27b has been shown to regulate HCV infection in the hepatocytes via various mechanisms that have not been fully elucidated. In this study, therefore, we examined the mechanisms of miR-27b-mediated regulation of HCV infection.

Methods: In silico screening analysis, transfection with miR-27b mimic, and a cell-based reporter assay were performed to identify miR-27b target genes. Cell cultured-derived HCV (HCVcc) was added to Huh7.5.1 cells knocked down for aquaporin (AQP) 11 (AQP11) and overexpressing AQP11. HCV replication levels were evaluated by real-time RT-PCR analysis of HCVcc genome.

Results: Infection of Huh7.5.1 cells with HCVcc resulted in significant elevation in miR-27b expression levels. In silico analysis revealed that AQP11, which is an AQP family member and is mainly localized in the endoplasmic reticulum (ER), was a candidate for a target gene of miR-27b. Transfection of a miR-27b mimic significantly reduced AQP11 expression, but a cell-based reporter assay demonstrated that miR-27b did not suppress the expression of a reporter gene containing the 3'-untranslated region of the AQP11 gene, suggesting that miR-27b indirectly suppressed AQP11 expression. AQP11 expression levels were significantly reduced by infection with HCVcc in Huh7.5.1 cells. Knockdown and over-expression of AQP11 significantly reduced and increased HCVcc genome levels in the cells following infection, respectively, however, AQP11 knockdown did not show significant effects on the HCVcc titers in the culture supernatants.

Conclusions: These results indicated that HCV infection induced a miR-27b-mediated reduction in AQP11 expression, leading to a modest reduction in HCV genome levels in the cells, not HCV titers in the culture supernatants.

Keywords: Aquaporin-11; HCV; miR-27b; microRNA.

Fig. 1

Increase in miR-27b expression following infection with HCVcc in Huh7.5.1 cells. a HCVcc genome copy numbers in Huh7.5.1 cells following infection. HCVcc genome copy numbers were normalized to GAPDH. b miR-27b expression levels in Huh7.5.1 cells following infection. Huh7.5.1 cells were infected with HCVcc at an MOI of 1. Total RNA was recovered from the cells at the indicated time points, followed by real-time RT-PCR analysis. miR-27b expression levels were normalized to U6. Expression levels relative to mock-infected cells are shown. c HCVcc genome copy numbers in Huh7.5.1 cells following transfection with miR-27b mimic. HCVcc genome copy numbers were determined by real-time PCR analysis 72 h after infection. HCVcc genome copy numbers were normalized to GAPDH. The relative HCVcc genome copy numbers in the cells transfected with 25 nM of control mimic were normalized to 1. The data are expressed as the means ± S.D. (N = 3). **p < 0.01, ***p < 0.001

Fig. 2

miR-27b-mediated suppression of AQP11 expression in Huh7.5.1 cells. a AQP11 mRNA levels in Huh7.5.1 cells following transfection with a miR-27b mimic. Huh7.5.1 cells were transfected with 25 nM of miR-27b mimic or control mimic. Following a 48-h incubation, total RNA was recovered from the cells, followed by real-time RT-PCR analysis. AQP11 mRNA levels were normalized to GAPDH. b AQP11 protein levels in Huh7.5.1 cells following transfection with a miR-27b mimic. Huh7.5.1 cells were transfected with 50 nM of miR-27b mimic or control mimic. Following a 72-h incubation, cell lysates were prepared, followed by western blot analysis. Representative image from two independent experiments are shown. c AQP11 mRNA levels in Huh7.5.1 cells following transfection with antagomiR-27b. Huh7.5.1 cells were transfected with 10 nM of antagomiR-27b or control antagomir. Following a 48-h incubation, total RNA was recovered, followed by real-time RT-PCR analysis. AQP11 mRNA levels were normalized to GAPDH. D, relative renilla luciferase activities following co-transfection with a reporter plasmid containing the 3′-UTR sequence of the AQP11 gene and miR-27b mimic. HEK293 cells were transfected with 25 nM of miR-27b mimic or control mimic and 1 μg/ml of reporter plasmids. Following a 72-h incubation, renilla and firefly luciferase activities were determined. Renilla luciferase activity levels were normalized to firefly luciferase activity. The data are expressed as the means ± S.D. (N = 3). *p < 0.05

Fig. 3

Reduction in AQP11 expression in Huh7.5.1 cells following infection with HCVcc. a AQP11 mRNA levels following infection with HCVcc at an MOI of 1. Total RNA was recovered on days 3 and 4, followed by real-time RT-PCR analysis. AQP11 mRNA levels were normalized to GAPDH. The relative AQP11 mRNA levels in mock-infected cells on each day were normalized to 1. The data are expressed as the means ± S.D. (N = 3). *p < 0.05. b AQP11 protein levels following infection with HCVcc. Huh7.5.1 cells were infected with HCVcc at an MOI of 1. Total cell lysates were prepared at the indicated time points, followed by western blot analysis. c AQP11 protein levels in HCV subgenomic replicon-expressing cells. Total cell lysates was prepared from Huh7.5.1 1b Feo cells and Huh7.5.1 cells, followed by western blot analysis. Representative western blot-analysis data from two independent experiments are shown

Fig. 4

AQP11 is a crucial factor for HCV genome replication. a knockdown efficiencies of siAQP11. AQP11 mRNA levels were determined 2 days after siRNA transfection. AQP11 mRNA levels were normalized to GAPDH. b HCVcc genome copy numbers in AQP11-knocked down cells. Huh7.5.1 cells were transfected with 50 nM of siAQP11. Two days after siRNA transfection, cells were infected with HCVcc at an MOI of 1. HCVcc genome copy numbers were determined 2 and 6 days after infection. HCVcc genome copy numbers were normalized to GAPDH. The relative HCV genome levels in siControl-treated cells on day 2 were normalized to 1. c cell viabilities following knockdown of AQP11. Huh7.5.1 cells were transfected with siAQP11. Cell viabilities were determined 3 days after transfection. The cell viabilities of the mock group were normalized to 100%. d AQP11 mRNA levels following transfection with pAQP11. AQP11 mRNA levels were determined 2 days after plasmid transfection. AQP11 mRNA levels were normalized to GAPDH. The relative AQP11 mRNA levels in pControl-treated cells were normalized to 1. e HCVcc genome copy numbers in AQP11-overexpressing cells. Huh7.5.1 cells were transfected with 6 μg/ml of pAQP11. Two days after plasmid transfection, cells were infected with HCVcc at an MOI of 0.5. HCVcc genome copy numbers were determined 4 days after infection. The HCVcc genome copy numbers were normalized to GAPDH. Relative HCV genome levels in pControl-treated cells were normalized to 1. f HCV subgenomic replicon levels in Huh7.5.1 1b Feo cells following AQP11 over-expression. Huh7.5.1 1b Feo cells were transfected with 6 μg/ml of pAQP11. HCV subgenomic replicon levels were determined 2 days after transfection. The data are expressed as the means ± S.D. (N = 3), *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

Model of miR-27b-mediated promotion of HCV replication via down-regulation of AQP11 expression. Following HCV infection, miR-27b expression is up-regulated, leading to indirect reduction in AQP11 expression and suppression of HCV genome replication