Epigenetic regulation of MicroRNA-122 by peroxisome proliferator activated receptor-gamma and hepatitis b virus X protein in hepatocellular carcinoma cells - PubMed (original) (raw)

. 2013 Nov;58(5):1681-92.

doi: 10.1002/hep.26514. Epub 2013 Sep 17.

Affiliations

Epigenetic regulation of MicroRNA-122 by peroxisome proliferator activated receptor-gamma and hepatitis b virus X protein in hepatocellular carcinoma cells

Kyoungsub Song et al. Hepatology. 2013 Nov.

Abstract

MicroRNA-122 (miR-122), a pivotal liver-specific miRNA, has been implicated in several liver diseases including hepatocellular carcinoma (HCC) and hepatitis C and B viral infection. This study aimed to explore epigenetic regulation of miR-122 in human HCC cells and to examine the effect of hepatitis C virus (HCV) and hepatitis B virus (HBV). We performed microRNA microarray analysis and identified miR-122 as the most up-regulated miRNA (6-fold) in human HCC cells treated with 5'aza-2'deoxycytidine (5-Aza-CdR, DNA methylation inhibitor) and 4-phenylbutyric acid (PBA, histone deacetylation inhibitor). Real-time polymerase chain reaction (PCR) analysis verified significant up-regulation of miR-122 by 5'aza and PBA in HCC cells, and to a lesser extent in primary hepatocytes. Peroxisome proliferator activated receptor-gamma (PPARγ) and retinoid X receptor alpha (RXRα) complex was found to be associated with the DR1 and DR2 consensus site in the miR-122 gene promoter which enhanced miR-122 gene transcription. 5-Aza-CdR and PBA treatment increased the association of PPARγ/RXRα, but decreased the association of its corepressors (N-CoR and SMRT), with the miR-122 DR1 and DR2 motifs. The aforementioned DNA-protein complex also contains SUV39H1, an H3K9 histone methyl transferase, which down-regulates miR-122 expression.

Conclusions: These findings establish a novel role of the PPARγ binding complex for epigenetic regulation of miR-122 in human HCC cells. Moreover, we show that hepatitis B virus X protein binds PPARγ and inhibits the transcription of miR-122, whereas hepatitis C viral particles exhibited no significant effect; these findings provide mechanistic insight into reduction of miR-122 in patients with HBV but not with HCV infection.

© 2013 by the American Association for the Study of Liver Diseases.

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Conflict of interest statement

No conflicts of interest exist

Figures

Figure 1

Figure 1. The expression of miR-122 is epigenetically suppressed in HCC cells

(A) MiRNA expression heat map depicting miRNAs differentially expressed (p<0.01) in HepG2 cells treated with control vehicle or with 5-Aza-CdR (3 μM) and PBA (3 mM) for 48 hours. (B) Summary of miRNA microarray data with at least 3 fold changes (compared to control) (p-value was calculated by ANOVA). (C) qRT-PCR analysis of mature miR-122 in HepG2 and Huh7 cells treated with control vehicle or with 5-Aza-CdR and PBA for 48 hours. The data were normalized to U6 RNA. (D) qRT-PCR analysis of pri-miR-122 in HepG2 and Huh7 cells treated with control vehicle or with 5-Aza-CdR and PBA for 48 hours. (E) qRT-PCR of miR-122 expression in HCC cell lines compared to human primary hepatocytes. (F) qRT-PCR of miR-122 expression in human primary hepatocytes treated with 5-Aza-CdR (3 μM) and PBA (3 mM) for 48 hours. The data represent mean + SD (***P<0.001, n = 3).

Figure 2

Figure 2. PPARγ/RXRα complex in 5-Aza-CdR/PBA-induced miR-122 expression

(A) 5-Aza-CdR/PBA induce the binding of endogenous PPARγ/RXRα to the DR1 and DR2 consensus site. (Upper panel) schematic representation of putative PPARγ/RXRα binding sites in human miR-122 gene promoter. (Mid panel) Equal amount of cell lysates from HepG2 cells were incubated with biotinylated double-stranded oligonucleotides corresponding to the DR1 and DR2 motifs in miR-122 promoter and with sterptavidin-agarose beads. The precipitated complexes were subjected to SDS-PAGE and Western blotting. (Lower panel) Western blot for PPARα in HepG2 cells with or without 5-Aza-CdR/PBA treatment. (B) 5-Aza-CdR/PBA induce PPARγ/RXRα binding to miR-122 DR1 and DR2 motifs in HepG2 cells with PPARγ overexpression. After transient transfection of PPARγ expression vector, the cells were treated with 5-Aza-CdR/PBA for 48 hours and the cell lysates were obtained for DNA pull down assay. (C) ChIP assay. The chromatin extracted from HepG2 cells treated with 5-Aza-CdR/PBA or control vehicle were subjected to immunoprecipitation with PPARγ antibody and the precipitates were subjected to qRT-PCR analysis using primers to amplify DR1 and DR2 regions as indicated in the schematic diagram (the arrowheads show the primer regions in the miR-122 promoter). Normal rabbit IgG was used as the negative control. (D) Effect of 5-Aza-CdR and PBA on miR-122 promoter luciferase activity in HepG2 and Huh7 cells. After transient transfection of miR-122-Luc promoter vectors, the cells were treated 5-Aza-CdR and PBA for 48 hours and the cell lysates were obtained for luciferase activity. (E) qRT-PCR for mature miR-122 in HepG2 cells treated with the PPARγ agonist (15-d-PGJ2, 15-keto-PGE2) or RXRα agonist (9-cis RA). The cells were transiently transfected with the PPARγ expression vector or control vector and the cells were incubated for 24 hours with DMSO or 10 μM agonist. qRT-PCR was performed to measure mature miR-122. (F) qRT-PCR for mature miR-122 in PPARγ overexpressed HepG2 cells treated with 10 μM of the PPARγ agonists (rosiglitazone, troglitazone and ciglitazone) or the vehicle control (DMSO). (G) qRT-PCR for mature miR-122 in NeHepLxHT cells transfected with the PPARγ siRNA or the PPARγ expression vector (Upper panel). Knockdown or overexpression of PPARγ in NeHepLxHT cells were confirmed by western blotting (Lower panel). The data represent mean + SD from (***P<0.001, n = 3).

Figure 3

Figure 3. 5-Aza-CdR/PBA-induced miR-122 expression is associated with N-CoR/SMRT corepressor dissociation and inhibition of SUV39H1

(A) Binding of endogenous N-CoR and SMRT to miR-122 DR1 and DR2 motifs in HepG2 cells treated with control vehicle or with 5-Aza-CdR/PBA. (B) Effect of 5-Aza-CdR and PBA on PPARγ association with N-CoR, SMRT and SUV39H1 in HepG2 cells. (Left panel) The cell lysates were immunoprecipitated with anti-PPARγ antibody followed by immunoblotting with indicated antibodies. (Right panel) Conventional western blotting using indicated antibodies. (C) Effect of 5-Aza-CdR and PBA on SUV39H1 expression in HepG2 and Huh7 cells. The cells were treated with 5-Aza-CdR/PBA for 48 hours and the cell lysates were obtained for Western blotting with anti-SUV39H1 antibody. (D) Binding of SUV39H1 to miR-122 DR1 and DR2 motifs. HepG2 cells were treated with 5-Aza-CdR and PBA for 48 hours and the cell lysates were incubated with biotinylated DR1 and DR2 oligonucleotides. The samples were subjected to western blotting using anti-SUV39H1. (E) The effect of SUV39H1 knockdown on miR-122 expression. HepG2 cells were transfected with two different siRNAs targeting SUV39H1 (100 nM) or mock siRNA as control. The efficiency of SUV39H1 knock-down was analyzed by western blotting 72 hours after transfection (left panel). qRT-PCR for mature miR-122 was performed (right panel). (F) The SUV39H1 inhibitor, chaetocin, increased miR-122 expression in HepG2 and Huh7 cells. The cells were treated with 200 nM chaetocin for 48 hours and qRT-PCR was performed to determine the level of miR-122. (G) Effect of 5-Aza-CdR/PBA on histone acetylation (chromatin immunoprecipitation assay). Chromatins extracted from HepG2 cells treated with 5-Aza-CdR/PBA or control vehicle were subjected to immunoprecipitation with anti-acetyl histone antibody. The precipitates were subjected to qRT-PCR analysis using primers to amplify DR1 and DR2 regions of the miR-122 gene promoter as indicated in the schematic diagram. The data represent mean + SD (***P<0.001, **P<0.01; n = 3).

Figure 4

Figure 4. Effect of HCV on miR-122 expression

(A) Huh7.5 cells were infected with JFH1-GFP-HCV virus for 96 hours. (Upper panel) Fluorescence microscopy showing intracellular expression of GFP in cells infected with JFH1-GFP-HCV. (Lower panel) qRT-PCR analysis for HCV RNA in cells with or without JFH1-GFP-HCV infection. (B) qRT-PCR analysis for miR-122 in Huh7.5 cells with or without JFH1-GFP-HCV infection for 96 hours. The results were normalized to the level of U6 RNA. (C) miR-122 promoter luciferase reporter activity in Huh7.5 cells with or without JFH1-GFP-HCV infection for 96 hours.

Figure 5

Figure 5. Effect of HBV on miR-122 expression

(A) HepG2.2.15 cells were cultured for 72 hours; DNA was extracted from HepG2.2.15 cells or supernatants (400 μl). HBV DNA was detected by qRT-PCR using HBV-specific primers (Left panel). HepG2 cells and their supernatants were used as negative controls. qRT-PCR analysis of mature miR-122 was performed in HepG2 and HepG2.2.15 cells (Right panel). (B) HepaRG cells were infected with HBV inoculums for 20 hours and incubated for 7 days. The medium was changed every 2 days. At the end of incubation, HBV DNAs were detected in the culture medium and HBV-infected cells (Left panel). Total RNAs extracted from uninfected or HBV-infected HepaRG cells were subjected to qRT-PCR analysis for miR-122 (Right panel). (C) Human primary hepatocytes were infected with HBV inoculums for 5 or 7 days. The HBX mRNA levels (Left panel) and miR-122 expression levels (Right panel) were determined by qRT-PCR. Levels of GAPDH mRNA and U6 were used as an internal control. The data represent mean + SD (***P<0.001, **P<0.01; n = 3).

Figure 6

Figure 6. Effect of HBX on miR-122 expression

(A) qRT-PCR analysis of miR-122 in HepG2 and Huh7 cells 48 hours after transfection with HBX expression vector or control vector (left and mid panel). Human primary hepatocytes were transfected with HBX for 72 hours and miR-122 expression levels were measured by qRT-PCR (right panel). (B) miR-122 promoter luciferase reporter activity in HepG2 and Huh7 cells 48 hours after transfection with HBX expression vector or control vector (***p<0.001). (C) Mock-siRNA and HBX-siRNA (100 nM) were transfected into HepG2.2.15 cells using lipofectamine. The levels of HBX protein (Left-upper panel) or mRNA (Left-lower panel) were detected by western blot and qRT-PCR (72 hours after transfection). The levels of miR-122 expression were measured by qRT-PCR in control and HBX-siRNA transfected HepG2.2.15 cells (Right panel). (D) HBX associates with PPARγ. HepG2 cells were transfected with the HBX expression vector or control vector and the cell lysates were immunoprecipitated with anti-PPARγ antibody followed by immunoblotting with anti-HBX antibody (upper panel). Conventional western blotting for HBX and β-actin in HBX transfected or vector control cells are shown at the lower panel. (E) qRT-PCR analysis for mature miR-122 in HepG2 cells transfected with HBX and/or PPARγ. The data represent mean + SD (***P<0.001, n = 3). (F) qRT-PCR analysis for pri-miR-122 in HepG2 cells transfected with HBX and/or PPARγ.

Figure 7

Figure 7. Schematic illustration of mechanisms for epigenetic regulation of miR-122

Epigenetic regulation of miR-122 in HCC cells and hepatocytes involves the PPARγ/RXRα/N-CoR/SMRT/SUV39H1/DR1/DR2 binding complex, histone acetylation, and histone H3K9 methylation. 5-Aza-CdR and PBA treatment inhibit SUV39H1 expression and cause dissociation of the corepressor complex (N-CoR/SMRT/SUV39H1) from PPARγ/RXRα/DR1/DR2, thus allowing PPARγ/RXRα-induced miR-122 gene transcription. Hepatitis B viral X protein inhibits PPARγ through direct binding and thus down-regulate miR-122 expression.

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