Hepatitis C virus infection and hepatic stellate cell activation downregulate miR-29: miR-29 overexpression reduces hepatitis C viral abundance in culture

Abstract

Background: Chronic hepatitis C virus (HCV)-induced liver fibrosis involves upregulation of transforming growth factor (TGF)-β and subsequent hepatic stellate cell (HSC) activation. MicroRNAs (miRNAs) regulate HCV infection and HSC activation.

Methods: TaqMan miRNA profiling identified 12 miRNA families differentially expressed between chronically HCV-infected human livers and uninfected controls. To identify pathways affected by miRNAs, we developed a new algorithm (pathway analysis of conserved targets), based on the probability of conserved targeting.

Results: This analysis suggested a role for miR-29 during HCV infection. Of interest, miR-29 was downregulated in most HCV-infected patients. miR-29 regulates expression of extracellular matrix proteins. In culture, HCV infection downregulated miR-29, and miR-29 overexpression reduced HCV RNA abundance. miR-29 also appears to play a role in HSCs. Hepatocytes and HSCs contribute similar amounts of miR-29 to whole liver. Both activation of primary HSCs and TGF-β treatment of immortalized HSCs downregulated miR-29. miR-29 overexpression in LX-2 cells decreased collagen expression and modestly decreased proliferation. miR-29 downregulation by HCV may derepress extracellular matrix synthesis during HSC activation.

Conclusions: HCV infection downregulates miR-29 in hepatocytes and may potentiate collagen synthesis by reducing miR-29 levels in activated HSCs. Treatment with miR-29 mimics in vivo might inhibit HCV while reducing fibrosis.

Figure 1.

Downregulation of miR-29 in C-HCV. Fold change in mature miR-29 levels relative to control uninfected livers. A, miR-29 levels in C-HCV patients measured by multiplex qRT TaqMan miRNA profiling. B, Individual qRT-PCR validated miR-29 downregulation in HCV-infected needle biopsies (Pearson's correlation between A and B: miR-29a, r = .653 P = .0010; miR-29b, r = .623 P = .0019; miR-29c, r = .752 P < .0001). Mean ± standard error of the mean (s.e.m.); three independent measurements. (C-G) HCVcc infection reduces miR-29, and miR-29 inhibits HCV. Fold change of (C), miR-29a, (D) miR-29b and (E) miR-29c in HCVcc-infected cells relative to mock-infected cells. Average of two independent experiments in triplicate. Mean ± s.e.m. (F) Huh7.5 cells were treated with miR-29 mimic or negative control mimic (control-pre-miR) and infected with HCVcc. Fold change in HCV RNA relative to control-pre-miR. Representative experiment from three independent experiments in triplicate. Mean ± s.e.m. (G) Cytotox-Fluor cytotoxicity assay. Positive control, 30 μg/ml digitonin. Average of three independent experiments in triplicate. Mean ± s.e.m. * indicates a P value < .05 for all panels.

Figure 2.

miR-29 downregulation in activated HSCs. Primary rat HSCs were isolated and activated in culture by plating on plastic. Data from three independent HSC isolations are shown (Time courses # 1, 2, and 3). Levels of (A) miR-29a, (B) miR-29b, and (C) miR-29c.

Figure 3.

miR-29 downregulation in TGF-β-treated HSCs but not in hepatocytes. miR-29 levels in (A) LX-2 HSC cell line or (B) Huh7.5 cells treated with TGF-β (10 ng/ml) for 24 hours. Representative experiment from two independent experiments in quadruplicate. Mean ± s.e.m. * indicates a P value < .05 for all panels.

Figure 4.

miR-29 overexpression in LX-2 cells inhibits proliferation and collagen expression. LX-2 cells were transfected with 25 nM miR-29 mimic or negative control mimic (control-pre-miR). A, WST-1 cell proliferation assay. Average of three independent experiments in quadruplicates. B, Ki67 mRNA levels. C, Col1A1, Col3A1, and α-SMA mRNA levels. Representative experiment from three independent experiments in triplicate. Mean ± s.e.m. D, Cytotox-Fluor cytotoxicity assay. Positive control, 30 μg/ml digitonin (E) Caspase-glow 3/7 apoptosis assay. Positive control, 2 μg/ml doxorubicin. Average of three independent experiments in quadruplicate. Mean ± s.e.m. * indicates a P-value < .05 for all panels.