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Modulation of HBV replication by microRNA-15b through targeting hepatocyte nuclear factor 1?.

ABSTRACT: Hepatitis B virus (HBV) infection remains a major health problem worldwide. The role played by microRNAs (miRNAs) in HBV replication and pathogenesis is being increasingly recognized. In this study, we found that miR-15b, an important miRNA during HBV infection and hepatocellular carcinoma development, directly binds hepatocyte nuclear factor 1? (HNF1?) mRNA, a negative regulator of HBV Enhancer I, to attenuate HNF1? expression, resulting in transactivation of HBV Enhancer I, in turn causing the enhancement of HBV replication and expression of HBV antigens, including HBx protein, finally leading to the down-regulated expression of miR-15b in both cell lines and mice in a long cascade of events. Our research showed that miR-15b promotes HBV replication by augmenting HBV Enhancer I activity via direct targeting HNF1?, while HBV replication and antigens expression, particularly the HBx protein, then repress the expression of miR-15b. The reciprocal regulation between miR-15b and HBV controls the level of HBV replication and might play a role in persistent HBV infection. This work adds to the body of knowledge concerning the complex interactions between HBV and host miRNAs.

SUBMITTER: Dai X

PROVIDER: S-EPMC4041434 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Modulation of HBV replication by microRNA-15b through targeting hepatocyte nuclear factor 1α.

Dai Xiaopeng X Zhang Wei W Zhang Hongfei H Sun Shihui S Yu Hong H Guo Yan Y Kou Zhihua Z Zhao Guangyu G Du Lanying L Jiang Shibo S Zhang Jianying J Li Junfeng J Zhou Yusen Y

Nucleic acids research 20140404 10

Hepatitis B virus (HBV) infection remains a major health problem worldwide. The role played by microRNAs (miRNAs) in HBV replication and pathogenesis is being increasingly recognized. In this study, we found that miR-15b, an important miRNA during HBV infection and hepatocellular carcinoma development, directly binds hepatocyte nuclear factor 1α (HNF1α) mRNA, a negative regulator of HBV Enhancer I, to attenuate HNF1α expression, resulting in transactivation of HBV Enhancer I, in turn causing the ...[more]

PMID: 24705650

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Project description:AIM:To uncover the role of hepatocyte nuclear factor 4 alpha (HNF4?) in regulating hepatic expression of microRNAs. METHODS:Microarray and real-time PCR were used to determine hepatic expression of microRNAs in young-adult mice lacking Hnf4? expression in liver (Hnf4?-LivKO). Integrative genomics viewer software was used to analyze the public chromatin immunoprecipitation-sequencing datasets for DNA-binding of HNF4?, RNA polymerase-II, and histone modifications to loci of microRNAs in mouse liver and human hepatoma cells. Dual-luciferase reporter assay was conducted to determine effects of HNF4? on the promoters of mouse and human microRNAs as well as effects of microRNAs on the untranslated regions (3'UTR) of two genes in human hepatoma cells. RESULTS:Microarray data indicated that most microRNAs remained unaltered by Hnf4? deficiency in Hnf4?-LivKO mice. However, certain liver-predominant microRNAs were down-regulated similarly in young-adult male and female Hnf4?-LivKO mice. The down-regulation of miR-101, miR-192, miR-193a, miR-194, miR-215, miR-802, and miR-122 as well as induction of miR-34 and miR-29 in male Hnf4?-LivKO mice were confirmed by real-time PCR. Analysis of public chromatin immunoprecipitation-sequencing data indicates that HNF4? directly binds to the promoters of miR-101, miR-122, miR-194-2/miR-192 and miR-193, which is associated with histone marks of active transcription. Luciferase reporter assay showed that HNF4? markedly activated the promoters of mouse and human miR-101b/miR-101-2 and the miR-194/miR-192 cluster. Additionally, miR-192 and miR-194 significantly decreased activities of luciferase reporters for the 3'UTR of histone H3F3 and chromodomain helicase DNA binding protein 1 (CHD1), respectively, suggesting that miR-192 and miR-194 might be important in chromosome remodeling through directly targeting H3F3 and CHD1. CONCLUSION:HNF4? is essential for hepatic basal expression of a group of liver-enriched microRNAs, including miR-101, miR-192, miR-193a, miR-194 and miR-802, through which HNF4? may play a major role in the post-transcriptional regulation of gene expression and maintenance of the epigenome in liver.

Project description:UnlabelledHepatitis B virus (HBV), a small enveloped DNA virus, chronically infects more than 350 million people worldwide and causes liver diseases from hepatitis to cirrhosis and liver cancer. Here, we report that hepatocyte nuclear factor 6 (HNF6), a liver-enriched transcription factor, can inhibit HBV gene expression and DNA replication. Overexpression of HNF6 inhibited, while knockdown of HNF6 expression enhanced, HBV gene expression and replication in hepatoma cells. Mechanistically, the SP2 promoter was inhibited by HNF6, which partly accounts for the inhibition on S mRNA. Detailed analysis showed that a cis element on the HBV genome (nucleotides [nt] 3009 to 3019) was responsible for the inhibition of the SP2 promoter by HNF6. Moreover, further analysis showed that HNF6 reduced viral pregenomic RNA (pgRNA) posttranscriptionally via accelerating the degradation of HBV pgRNA independent of La protein. Furthermore, by using truncated mutation experiments, we demonstrated that the N-terminal region of HNF6 was responsible for its inhibitory effects. Importantly, introduction of an HNF6 expression construct with the HBV genome into the mouse liver using hydrodynamic injection resulted in a significant reduction in viral gene expression and DNA replication. Overall, our data demonstrated that HNF6 is a novel host factor that can restrict HBV replication via both transcriptional and posttranscriptional mechanisms.ImportanceHBV is a major human pathogen whose replication is regulated by host factors. Liver-enriched transcription factors are critical for many liver functions, including metabolism, development, and cell proliferation, and some of them have been shown to regulate HBV gene expression or replication in different manners. In this study, we showed that HNF6 could inhibit the gene expression and DNA replication of HBV via both transcriptional and posttranscriptional mechanisms. As HNF6 is differentially expressed in men and women, the current results may suggest a role of HNF6 in the gender dimorphism of HBV infection.

Project description:BACKGROUND AND PURPOSE:Metformin, a small molecule, antihyperglycaemic agent, is a well-known activator of AMP-activated protein kinase (AMPK) and protects against cardiac fibrosis. However, the underlying mechanisms remain elusive. TGF?1 is a key cytokine mediating cardiac fibrosis. Here, we investigated the effects of metformin on TGF?1 production induced by angiotensin II (AngII) and the underlying mechanisms. EXPERIMENTAL APPROACH:Wild-type and AMPK?2-/- C57BL/6 mice were injected s.c. with metformin or saline and infused with AngII (3 mg·kg-1 ·day-1 ) for 7 days. Adult mouse cardiac fibroblasts (CFs) were isolated for in vitro experiments. KEY RESULTS:In CFs, metformin inhibited AngII-induced TGF?1 expression via AMPK activation. Analysis using bioinformatics predicted a potential hepatocyte nuclear factor 4? (HNF4?)-binding site in the promoter region of the Tgfb1 gene. Overexpressing HNF4? increased TGF?1 expression in CFs. HNF4? siRNA attenuated AngII-induced TGF?1 production and cardiac fibrosis in vitro and in vivo. Metformin inhibited the AngII-induced increases in HNF4? protein expression and binding to the Tgfb1 promoter in CFs. In vivo, metformin blocked the AngII-induced increase in cardiac HNF4? protein levels in wild-type mice but not in AMPK?2-/- mice. Consequently, metformin inhibited AngII-induced TGF?1 production and cardiac fibrosis in wild-type mice but not in AMPK?2-/- mice. CONCLUSIONS AND IMPLICATIONS:HNF4? mediates AngII-induced TGF?1 transcription and cardiac fibrosis. Metformin inhibits AngII-induced HNF4? expression via AMPK activation, thus decreasing TGF?1 transcription and cardiac fibrosis. These findings reveal a novel antifibrotic mechanism of action of metformin and identify HNF4? as a new potential therapeutic target for cardiac fibrosis. LINKED ARTICLES:This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.

Dataset Information

Modulation of HBV replication by microRNA-15b through targeting hepatocyte nuclear factor 1?.

Publications

Modulation of HBV replication by microRNA-15b through targeting hepatocyte nuclear factor 1α.

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