Upregulation of microRNA-146a by hepatitis B virus X protein contributes to hepatitis development by downregulating complement factor H - PubMed (original) (raw)
. 2015 Mar 24;6(2):e02459-14.
doi: 10.1128/mBio.02459-14.
Xiao-Peng Dai 1, Wei Zhang 1, Shi-Hui Sun 1, Yang Zeng 1, Guang-Yu Zhao 1, Zhi-Hua Kou 1, Yan Guo 1, Hong Yu 1, Lan-Ying Du 2, Shi-Bo Jiang, Yu-Sen Zhou 3
Affiliations
- PMID: 25805734
- PMCID: PMC4453536
- DOI: 10.1128/mBio.02459-14
Upregulation of microRNA-146a by hepatitis B virus X protein contributes to hepatitis development by downregulating complement factor H
Jun-Feng Li et al. mBio. 2015.
Abstract
Hepatic injuries in hepatitis B virus (HBV) patients are caused by immune responses of the host. In our previous study, microRNA-146a (miR-146a), an innate immunity-related miRNA, and complement factor H (CFH), an important negative regulator of the alternative pathway of complement activation, were differentially expressed in HBV-expressing and HBV-free hepatocytes. Here, the roles of these factors in HBV-related liver inflammation were analyzed in detail. The expression levels of miR-146a and CFH in HBV-expressing hepatocytes were assessed via analyses of hepatocyte cell lines, transgenic mice, adenovirus-infected mice, and HBV-positive human liver samples. The expression level of miR-146a was upregulated in HBV-expressing Huh-7 hepatocytes, HBV-expressing mice, and patients with HBV infection. Further results demonstrated that the HBV X protein (HBx) was responsible for its effects on miR-146a expression through NF-κB-mediated enhancement of miR-146a promoter activity. HBV/HBx also downregulated the expression of CFH mRNA in hepatocyte cell lines and the livers of humans and transgenic mice. Furthermore, overexpression and inhibition of miR-146a in Huh-7 cells downregulated and upregulated CFH mRNA levels, respectively. Luciferase reporter assays demonstrated that miR-146a downregulated CFH mRNA expression in hepatocytes via 3'-untranslated-region (UTR) pairing. The overall effect of this process in vivo is to promote liver inflammation. These results demonstrate that the HBx-miR-146a-CFH-complement activation regulation pathway might play an important role in the immunopathogenesis of chronic HBV infection. These findings have important implications for understanding the immunopathogenesis of chronic hepatitis B and developing effective therapeutic interventions.
Importance: Hepatitis B virus (HBV) remains an important pathogen and can cause severe liver diseases, including hepatitis, liver cirrhosis, and hepatocellular carcinoma. Although HBV was found in 1966, the molecular mechanisms of pathogenesis are still poorly understood. In the present study, we found that the HBV X protein (HBx) promoted the expression of miR-146a, an innate immunity-related miRNA, through the NF-κB signal pathway and that increasingly expressed miR-146a downregulated its target complement factor H (CFH), an important negative regulator of the complement alternative pathway, leading to the promotion of liver inflammation. We demonstrated that the HBx-miR-146a-CFH-complement activation regulation pathway is potentially an important mechanism of immunopathogenesis caused by chronic HBV infection. Our data provide a novel molecular mechanism of HBV pathogenesis and thus help to understand the correlations between the complement system, an important part of innate immunity, and HBV-associated disease. These findings will also be important to identify potential therapeutic targets for HBV infection.
Copyright © 2015 Li et al.
Figures
FIG 1
miR-146a is upregulated in HBV-replicating hepatocytes and livers of HBV-infected mice. (A) Quantitative RT-PCR analyses of the relative expression levels of endogenous miR-146a in HepG2, HepG2.2.15, and HepAD38 cells. Huh-7 cells transiently transfected with pHBV1.2 or a control vector (pCtr) (B) and liver tissues of C57BL/6 mice 7 days after receiving a hydrodynamic injection of AdGFP-HBV or AdGFP (C). Each assay was performed in triplicate, and the expression level of miR-146a was normalized to that of U6 snRNA. Data are presented as the means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001, versus control or as indicated.
FIG 2
HBx induces miR-146a expression by enhancing its promoter activity. (A and B) The activity of a luciferase reporter gene under the control of the miR-146a promoter in cells that were cotransfected with pGL-146aP and pHBV1.2 (A) or a pcDNA3.1-based vector expressing HBc (HBVCore), large surface protein (HBVLs), viral polymerase (HBVPol), or HBx (B). (C) Quantitative RT-PCR analyses of miR-146a expression in Huh-7 cells that were transiently transfected with pcDNA3.1-HBx or empty vector. The expression level of miR-146a was normalized to that of U6 snRNA. Data are presented as the means ± SEM from n = 3 replicates. **, P < 0.01; ***, P < 0.001, versus control.
FIG 3
HBx upregulates the activity of the miR-146a promoter through NF-κB. (A) The left panel shows a schematic illustration of the miR-146a promoter segments used to generate the PGL3 luciferase reporter constructs. The wild-type and mutated NF-κB binding sites are also shown. The right panel shows the luciferase activities of the PGL3 reporter constructs containing the miR-146a promoter regions. The assays were performed 48 h after transfection of Huh-7 cells. (B) Effect of transient overexpression of HBx on the activities of the miR-146a promoter reporter constructs shown in panel A; (C and D) effects of pyrrolidine dithiocarbamate (PDTC), an NF-κB signaling inhibitor, on the HBx-mediated increases in miR-146a promoter activity (C) and miR-146a expression (D) in transiently transfected Huh-7 cells. Data are presented as the means ± SEM from n = 3 replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001, versus control or as indicated.
FIG 4
HBx downregulates CFH expression in hepatocytes and hepatic tissues. (A to D) Quantitative RT-PCR analyses of CFH mRNA levels in HepG2, HepG2.2.15, and HepAD38 cells (A), Huh-7 cells that were transiently transfected with a control vector, pHBV1.2 (B) or pcDNA3.1-HBx (C), and liver tissues of 7-month-old wild-type (WT) mice and 7- and 16-month-old p21-HBx-transgenic mice (D). (E) Immunoblot analyses of CFH protein in liver tissues of WT and p21-HBx-transgenic mice. The expression level of GAPDH was used as the internal control. Data are presented as the means ± SEM from n = 3 replicates. *, P < 0.05; **, P < 0.01, versus control (A to C) or WT (D) mice.
FIG 5
HBx-mediated downregulation of CFH expression requires miR-146a. (A) The effect of overexpression of HBx on the activity of a reporter gene under the control of the CFH 3' UTR. Dual-luciferase reporter assays of Huh-7 cells that were cotransfected with pcDNA3.1 or pcDNA3.1-HBx and pMIR or pMIR-CFH3'UTR were performed 48 h after transfection. Quantitative RT-PCR (B) and immunoblot analyses (C) of the effect of transient overexpression of miR-146a (pIRES-EGFP-146a) on CFH mRNA levels in Huh-7 cells. The effects of overexpression (pIRES-EGFP-146a) (C) or inhibition (pSD14-146a) (D) of miR-146a, or overexpression of HBx (pcDNA3.1-HBx) with or without inhibition of miR-146a (E and F), on the activity of pMIR-CFH3' UTR or pMIR-CFH3' UTR-M in transiently cotransfected Huh-7 cells. The reporter activities were determined at 48 h posttransfection. (A to D) Data are presented as the means ± SEM from n = 3 replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 6
Reduced CFH expression is associated with HBV/HBx-induced liver inflammation. (A) Quantitative RT-PCR of the expression levels of CFH mRNA in C57BL/6 mice infected with AdGFP-HBx, AdGFP-HBV, AdGFP, or phosphate-buffered saline (PBS) via hydrodynamic injection; (B) immunoblot analyses of CFH protein in C57BL/6 mice infected with AdGFP or AdGFP-HBV; (C) the serum levels of ALT and AST in C57BL/6 mice infected with AdGFP, AdGFP-HBx, or AdGFP-HBV; (D) hematoxylin- and eosin-stained sections of the livers of C57BL/6 and C3−/− mice infected with the indicated adenoviruses or PBS. The mice were euthanized 7 days after the injection. Data are presented as the means ± SEM from n = 5 replicates. **, P < 0.01. Scale bar, 100 µm.
FIG 7
miR-146a and CFH are up- and downregulated, respectively, in HBV-positive cirrhotic human liver tissues. (A and B) The relative miR-146a (A) and CFH mRNA (B) expression levels in liver tissues from HBV-positive liver transplant patients with cirrhosis (n = 9) and healthy transplant donors (n = 8). Data are presented as the geometric mean with 95% CI. ***, P < 0.001.
FIG 8
Overview of the regulation pathway from HBV infection to complement activation. HBx/HBV and several other viruses, such as Epstein-Barr virus (EBV), human T-cell lymphotropic virus 1 (HTLV-1), and vesicular stomatitis virus (VSV), as well as immune activators such as lipopolysaccharide (LPS) and interleukin 1β (IL-1β), can induce miR-146a expression by activating NF-κB signaling. miR-146a suppresses the expression of CFH, as well as multiple other genes, by binding to the 3' UTRs of its targets. Together with several other negative regulators of complement activation, CFH inhibits the formation of C3 convertase (C3bBb), thus inhibiting constitutive complement activation via the alternative pathway. HBx may also enhance the expression of CD59 and CD46, while HBc may inhibit CD59 expression. The HBx–miR-146a–CFH–complement activation pathway could play a central role in chronic HBV infection-related liver inflammation.
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