Epigenetic regulation of miR-34a expression in alcoholic liver injury - PubMed (original) (raw)

. 2012 Sep;181(3):804-17.

doi: 10.1016/j.ajpath.2012.06.010. Epub 2012 Jul 25.

Shannon S Glaser, Heather Francis, Fuquan Yang, Yuyan Han, Allison Stokes, Dustin Staloch, Jennifer McCarra, Jingang Liu, Julie Venter, Haiying Zhao, Xiuping Liu, Taylor Francis, Scott Swendsen, Chang-Gong Liu, Hidekazu Tsukamoto, Gianfranco Alpini

Affiliations

Epigenetic regulation of miR-34a expression in alcoholic liver injury

Fanyin Meng et al. Am J Pathol. 2012 Sep.

Abstract

Epigenetic changes are associated with the regulation of transcription of key cell regulatory genes [micro RNAs (miRNAs)] during different types of liver injury. This study evaluated the role of methylation-associated miRNA, miR-34a, in alcoholic liver diseases. We identified that ethanol feeding for 4 weeks significantly up-regulated 0.8% of known miRNA compared with controls, including miR-34a. Treatment of normal human hepatocytes (N-Heps) and cholangiocytes [human intrahepatic biliary epithelial cells (HiBECs)] with ethanol and lipopolysaccharide induced a significant increase of miR-34a expression. Overexpression of miR-34a decreased ethanol-induced apoptosis in both N-Heps and HiBECs. In support of the concept that the 5'-promoter region of miR-34a was noted to be embedded within a CpG island, the expression level of miR-34a was significantly increased after demethylation treatment in N-Heps and HiBECs. By methylation-specific PCR, we confirmed that miR-34a activation is associated with ethanol-linked hypomethylation of the miR-34a promoter. A combination of bioinformatics, dual-luciferase reporter assay, mass spectrometry, and Western blot analysis revealed that caspase-2 and sirtuin 1 are the direct targets of miR-34a. Furthermore, modulation of miR-34a also altered expression of matrix metalloproteases 1 and 2, the mediators involved in hepatic remodeling during alcoholic liver fibrosis. These findings provide the basis for an exciting field in which the epigenomic microRNAs of hepatic cells may be manipulated with potential therapeutic benefits in human alcoholic liver diseases.

Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1

Aberrant miR-34a expression in ethanol-exposed mouse liver. A: Left: Liver H&E staining of control (top) and ethanol-exposed (bottom) mouse liver that showed zonation of the liver, including portal and central veins, and miRNA expression patterns (middle) in ethanol-exposed mouse liver samples analyzed using a self-organizing tree algorithm using the Multiexperiment Viewer version 3.1 from the Institute for Genomic Research (Rockville, MD). A dendrogram showing three clusters was generated. miRNA expression in ethanol-exposed mouse liver tissues is shown on the right axis relative to normal mouse liver controls on the left axis. Cluster 1 is composed of a group of miRNAs, including miR-34a and miR-21, that were overexpressed after long-term alcohol exposure; cluster 2, 245 miRNAs that were not significantly altered after ethanol exposure; and cluster 3, 107 miRNAs that were decreased in expression. Right: Relative miRNA expression profile between ethanol-exposed livers versus normal control tissues. The expression of a panel of diverse updated miRNAs was evaluated by microarray analysis using Affymetrix GeneChip U133 Plus version 2.0 (Affymetrix Corporation, Santa Clara, CA). miRNA expression relative to U6 RNA was plotted, depicting the relative expression levels (Log2) for selected miRNAs in ethanol-exposed liver versus normal control panels (more than twofold change; P < 0.05). The relative expression levels and P values for each miRNA in the related samples were plotted against each other in the scatterplot. miR-34a and miR-122 are the most up- and down-regulated miRNAs among the 407 miRNAs detected in mice liver. Data represent the mean of three separate experiments. B and C: miRNA was isolated from either normal or ethanol-exposed mice liver (B) or from N-Heps, HiBECs, and HepG2 cell lines (C) with or without ethanol treatment (100 mmol/L) for 72 hours. The expression of a selected miRNA from each cluster (miR-34a from cluster 1, miR-96 from cluster 2, and miR-122a from cluster 3) was assessed using Taqman real-time PCR assay. Results represent the mean ± SE of miRNA expression from four separate determinations. D: Ethanol reduces hepatobiliary cell survival. Cell survival was assessed using a viable cell assay, and the survival index was assessed after the treatment of ethanol (100 mmol/L) or PBS for 72 hours. E: miR-34a is increased in human ALD liver. Total RNA was isolated from liver from control (C) or long-term ethanol-exposed patients (E). Real-time PCR analysis was performed, and the ratio of miR-34a to U6 small RNA expression in ethanol-exposed liver samples was determined. The PCR products were verified by 1.8% agarose gel electrophoresis. Data represent mean ± SE from three separate experiments. *P < 0.05 relative to controls.

Figure 2

Figure 2

Epigenetic regulation of miR-34a in ethanol-exposed mouse liver. A: The expression levels of DNMT1 and DNMT3B are down-regulated in ethanol-exposed mouse liver. Relative gene expression profile between ethanol-exposed mouse livers versus control liver tissues is shown. The expression of a panel of diverse epigenetic-associated genes was evaluated by real-time PCR using Mouse Epigenetic Chromatin Modification Enzymes PCR Array (PAMM-085A) from SABiosciences Corporation (Valencia, CA). Gene expression relative to GAPDH was plotted as the Volcano Plots, depicting the relative expression levels (Log10) for selected genes in ALD versus control panels (left). The relative expression levels and P values for each gene in the related samples were also plotted against each other in the scatterplot (right). DNMT1 and DNMT3B are the greatest down-regulated genes among the six epigenetic signaling pathways in ethanol-exposed mouse liver. Data represent mean from three separate experiments. B: RT-PCR confirmed the reduced mRNA expression of DNMT1 and DNMT3B in ethanol-exposed mouse liver relative to controls. Expression of DNMTs was assayed by a standard RT-PCR method in control and ethanol-exposed mouse liver tissues. The PCR amplification was performed for 26 cycles for GAPDH (a housekeeping control), 30 cycles for DNMT1, and 35 cycles for DNMT3B. The ratios shown represent the mean value (relative to control) normalized with GAPDH from four independent experiments. C and D: Methylation-specific PCR analysis of the miR-34a upstream regulatory region was performed in ethanol-exposed mice and normal control liver, as well as ethanol-treated N-Heps and HepG2 cells (100 mmol/L, 7 days). The dark gray bar indicates hypermethylated miR-34a; the white bar indicates unmethylated miR-34a. Long-term alcohol exposure significantly demethylated CpG island enriched 5′-promoter region of miR-34a. miR-34a promoter was also hypomethylated in N-Heps but not in HepG2 cells after ethanol treatment. The results shown represent the mean ± SE from six independent experiments. **P < 0.01 compared with expression in control regions. E and F: Characterization of miR-34a promoter methylation activities by mutation site-specific luciferase assay. Experimental design strategy of miR-34a methylation-specific mutation is presented (E). A 24-bp mutation of the hsa-miR-34a methylation site (106 bp) converted from GCC to TAA was performed using oligonucleotides to the CpG island enriched region. Luciferase reporter constructs containing the sequence of potential methylation site (CpG island enriched region) from the 5′-promoter region of miR-34a inserted downstream of the luciferase gene were generated. miR-34a-LUC (wild type) contains the intact sequence whereas miR-34a-MUT contained the sequence with GCC to TAA nucleotide changes. Reporter constructs were transfected in normal human hepatocytes for 24 hours and then treated with 10 μmol/L 5-Aza-CdR or diluent control for 72 hours (F). The expression of firefly luciferase activity was normalized to that of Renilla luciferase activity for each sample. The increases in relative firefly luciferase activity in the presence of methylation inhibitor in miR-34a-LUC but not in miR-34a-MUT transfected cells indicate the presence of a methylation-modulated target sequence in the 5′-promoter region of miR-34a. The results shown represent the mean ± SE from eight independent experiments. G: N-Heps, HiBECs, and HepG2 cells were treated with 10 μmol/L 5-Aza-CdR for 72 hours. The expression of mature miR-34a, miR-96, and miR-122 was assessed using Taqman real-time PCR assay. 5-Aza-CdR increased miR-34a but not miR-96 expression in normal hepatocytes and cholangiocytes. Data represent mean ± SE from eight separate experiments. H: 5-Aza-CdR treated hepatobiliary cells were subjected to ethanol treatment for 72 hours simultaneously. Cell viability was measured by the MTS assay. Demethylation treatment significantly enhanced cell survival against ethanol in N-Heps and HiBECs but not HepG2 HCC cells. The results shown represent the mean ± SE from four independent experiments. *P < 0.05 relative to controls. NS, no significant difference.

Figure 3

Figure 3

Overexpression of miR-34a increases cell survival and migration. A: miR-34a expression was assessed by real-time PCR in hepatobiliary cells transfected with either control or miR-34a precursors. The ability of these constructs to modulate miR-34a expression was verified in all three cell lines. Data represent mean ± SE from four separate experiments. B and C: Hepatobiliary cells were transfected with pre-miR-34a or control precursor. B: Cells were plated in agar wells in 96-well plates, and anchorage-independent growth assessed fluorometrically after 7 days. Pre-miR-34a increased anchorage independent growth in all three cell lines tested. The results shown represent the mean ± SE of four independent experiments. C: Cell survival against ethanol treatment (100 mmol/L for 72 hours) was assessed using a viable cell assay, and the survival index was assessed after 72 hours. Pre-miR-34a increased survival rate in cell lines from N-Heps and HiBECs but not from HepG2 cells. Data represent mean ± SE from four separate experiments. D: The hepatobiliary cells were transfected with either control or anti-miR-34a inhibitor for 48 hours. The transfected cells along with untransfected controls were plated in 96-well plates and treated with 20 mmol/L ethanol for 7 days. The proliferation index was measured by MTS assay in untransfected and transfected cells with or without ethanol treatment as indicted. Silencing of miR-34a has significantly further reduced cell survival in ethanol-treated human hepatocytes and HiBECs. Data represent mean ± SE from four separate experiments. E and F: N-Heps and HiBECs were transfected with either control or pre-miR-34a inhibitor for 48 hours. The transfected cells along with untransfected controls were further treated with 20 mmol/L ethanol for 7 days. Relative miR-34a expression (E) and methylation status of miR-34a upstream regulatory region (F) were detected by real-time PCR analysis, respectively. Ethanol-induced miR-34a up-regulation is more apparent in untransfected cells than that in miR-34a transfected cells (E). Furthermore, the degree of methylation is not altered in nontransfected hepatic cells compared with miR-34a transfected cell (both with ethanol treatment). The results shown represent the mean ± SE from four independent experiments. G: Cell migration was assessed as described in Materials and Methods and is expressed as arbitrary fluorescence units (AFU). The cell lines varied in their ability to migrate. Pre-miR-34a increased cell motility in all three cell lines. The results shown represent the mean ± SE from four independent experiments. *P < 0.05, **P < 0.01 relative to controls. †P < 0.05 relative to control miRNA precursor/inhibitor transfected cells.

Figure 4

Figure 4

Ethanol and DNMT3B silencing alter cell migration, survival, and transformation. A and B: N-Heps and HiBECs were treated with 20 mmol/L ethanol for 7 days. Cell migration (A) and transformation (B) were detected in treated cells by migration and soft agar assay. Relative longer-term ethanol treatment significantly increased cell motility and transformed survival potential in hepatobiliary cells tested. Data represent mean ± SE from six separate experiments. C and D: Cell survival against ethanol (C) and transformed cell growth (D) was assessed by MTS and soft agar assay in hepatobiliary cells transfected with either control or DNMT3B small-interfering RNA. Silencing DNMT3B increased the survival in normal hepatocytes but reduced HCC cell survival (C). In contrast to miR-34a, it decreased transformed cell growth in HepG2 cells (D). Data represent mean ± SE of eight separate experiments. *P < 0.05 relative to controls.

Figure 5

Figure 5

miR-34a regulates expression of SIRT1. A: Identification of protein targets modulated by miR-34a in N-Heps by HPLC-Chip/MS analysis. N-Heps were transfected with pre-miR-34a or control precursor (100 nmol/L) for 72 hours; HPLC-Chip/MS analysis was performed in protein lysates. Overexpressed or down-regulated proteins (more than twofold or <0.5-fold) from LC-MS have been ranked to their relative expression levels. Multifunctional oncogenic protein β-catenin is ranked in the up-regulated protein group, whereas CASP2 and SIRT1 are listed among the down-regulated targets. Data represent mean from three separate experiments. B: miR-34a target proteins are altered in normal hepatocytes after ethanol treatment. Hepatobiliary cells were treated with ethanol (100 mmol/L) or PBS controls. Cell lysates were obtained after 7 days and Western blots performed for CASP2, SIRT1, and α-tubulin. Treatment with ethanol down-regulated the expression of CASP2 and SIRT1 in all three cell lines. C and D: Schematic of predicted miR-34a site in the 3′UTR of human CASP2 and SIRT1. Positions 2 to 9 of the 5′ region of miRNA 34a are labeled in red. E and F: Luciferase reporter constructs containing the miR-34a recognition sequence from the 3′-UTR of CASP2 and SIRT1 inserted downstream of the luciferase gene were generated. pMIR-CASP2-wt-luc or pMIR-SIRT1-wt-luc contains the intact sequence, whereas pMIR-CASP2-mut-luc or pMIR-SIRT1-mut-luc contained the sequence with random nucleotide changes. Reporter constructs were co-transfected with either miR-34a precursor or control precursor in normal human hepatocytes. The expression of firefly luciferase activity was normalized to that of Renilla luciferase activity for each sample. The decreases in relative firefly luciferase activity in the presence of miR-34a indicate the presence of a miR-34a modulated target sequence in the 3′-UTR of CASP2 and SIRT1. Data represent the mean of eight separate experiments. *P < 0.05 relative to control precursor group. G: N-Heps were transfected with pre-miR-34a or control precursor. Cell lysates were obtained after 48 hours and Western blots performed for CASP2, SIRT1, survivin, and α-tubulin. Relative ratios normalized with α-tubulin and control group were displayed under the image. Overexpression of miR-34a decreased the expression of CASP2 and SIRT1 and subsequently increased the level of survivin, a downstream mediator of SIRT1 in N-Heps. The ratios shown represent the mean value (relative to control) normalized with α-tubulin from three independent experiments.

Figure 6

Figure 6

miR-34a regulates the tissue remodeling molecules during ethanol exposure. A and B: Expression of MMP-1, MMP-2, and MMP-9 mRNA was assessed by quantitative real-time PCR and normalized to expression of β-actin in normal liver and ethanol-exposed mouse tissue (A) and in normal hepatocytes with or without 7 days of ethanol treatment (B). MMP-1, MMP-2, and MMP-9 are overexpressed in ethanol-exposed liver compared with normal liver. However, MMP-2 and MMP-9, but not MMP-1, were increased in ethanol-treated hepatocytes compared with PBS controls. The results shown represent the mea ± SE of four independent experiments. C: N-Heps were transfected with miR-34a precursors or controls. After 72 hours, MMP Zymogen Gel Assay was performed for MMP-1, MMP-2, and MMP-9 expression. Enhanced expression of miR-34a in N-Heps increases MMP-2 and MMP-9 expression. D: Steatohepatitis is induced by 4 weeks of intragastric ethanol infusion (ISI) in mice liver. Liver histologic analysis of regular feeding control mice and ISI mice is displayed. Enhanced expression of MMP-9 and reduced expression of CASP2 and SIRT1 were seen in ethanol-exposed mouse liver [H&E; original magnification: ×40 (top); ×200 (bottom)]. E and F: Liver tissue homogenates were obtained from ethanol-exposed and control mice. Increases of liver fibrotic marker α-SMA and MMP-9 along with the reduction of CASP2 and SIRT1 were verified by Western blot analysis. Representative immunoblots (E) and quantitative data (mean ± SE) from four separate blots (F) are shown.

Figure 7

Figure 7

Summary diagram. Ethanol modulates liver parenchymal cell survival, remodeling, and transformation by activation of miR-34a–dependent signaling pathways.

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