Epigenetic Regulation of miR-34a Expression in Alcoholic Liver Injury
2012; Elsevier BV; Volume: 181; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2012.06.010
ISSN1525-2191
AutoresFanyin Meng, Shannon Glaser, Heather Francis, Fuquan Yang, Yuyan Han, Allison Stokes, Dustin Staloch, Jennifer McCarra, Jin‐Gang Liu, Julie Venter, Haiying Zhao, Xiuping Liu, Taylor Francis, Scott Swendsen, Chang‐Gong Liu, Hidekazu Tsukamoto, Gianfranco Alpini,
Tópico(s)Circular RNAs in diseases
ResumoEpigenetic 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. 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. Long-term alcohol consumption and the associated development of alcoholic liver disease (ALD) is a major health concern for the United States. Approximately 15% of individuals with alcoholism in the United States eventually develop ALD, one of the leading causes of liver diseases and liver-related deaths worldwide. ALDs encompass a broad spectrum of clinical features of alcoholic fatty liver, alcoholic steatohepatitis, alcoholic cirrhosis, and increased risk of hepatocellular carcinoma (HCC).1Mathurin P. Louvet A. Dharancy S. [Acute alcoholic hepatitis: management practices for 2007] French.Gastroenterol Clin Biol. 2008; 32: S179-S181Crossref PubMed Scopus (3) Google Scholar The pathologic mechanisms of ALD involve complex interactions between the direct effects of alcohol and its toxic metabolites on various cell types in the liver, including induction of reactive oxygen species, up-regulation of the inflammatory cascade, and other cell-specific effects in the liver. Prominent features of ALD include ethanol-mediated cellular alterations, steatosis, and hepatic inflammation. However, a comprehensive understanding of the mechanisms involved in the pathogenesis of ALD remains incomplete. Thus, an understanding of the molecular mechanisms regulating hepatobiliary cell injury is important and may lead to more effective therapeutic approaches for ALD. MicroRNAs (miRNAs) are a group of noncoding RNA that plays an important role in human liver diseases and have recently become of interest in the pathogenesis of ALD.2Lakner A.M. Bonkovsky H.L. Schrum L.W. microRNAs: fad or future of liver disease.World J Gastroenterol. 2011; 17: 2536-2542Crossref PubMed Scopus (67) Google Scholar, 3Kerr T.A. Korenblat K.M. Davidson N.O. MicroRNAs and liver disease.Transl Res. 2011; 157: 241-252Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar In mammals, miRNAs can negatively regulate their targets by either binding to imperfect complementary sites within the 3′-untranslated region (UTR) of their mRNA targets or by targeting specific cleavage of homologous mRNAs.4Bartel D.P. MicroRNAs: genomics, biogenesis, mechanism, and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (28615) Google Scholar In our previous studies, we observed the increased expression of several miRNAs, including miR-181 and let-7 family members that are involved in hepatic cell survival, remodeling, and transformation.5Meng F. Glaser S.S. Francis H. Demorrow S. Han Y. Passarini J.D. Stokes A. Cleary J.P. Liu X. Venter J. Kumar P. Priester S. Hubble L. Stoloch D. Sharma J. Liu C.G. Alpini G. Functional analysis of microRNAs in human hepatocellular cancer stem cells.J Cell Mol Med. 2012; 16: 160-173Crossref PubMed Scopus (104) Google Scholar Similarly, altered expression of several miRNAs has been described in expression profiling of human liver diseases and in animal studies.6Roderburg C. Urban G.W. Bettermann K. Vucur M. Zimmermann H. Schmidt S. Janssen J. Koppe C. Knolle P. Castoldi M. Tacke F. Trautwein C. Luedde T. 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In fact, aberrant expression of miRNAs, such as miR-181, alters the cellular expression of TIMP3 and Nemo-like kinase.5Meng F. Glaser S.S. Francis H. Demorrow S. Han Y. Passarini J.D. Stokes A. Cleary J.P. Liu X. Venter J. Kumar P. Priester S. Hubble L. Stoloch D. Sharma J. Liu C.G. Alpini G. Functional analysis of microRNAs in human hepatocellular cancer stem cells.J Cell Mol Med. 2012; 16: 160-173Crossref PubMed Scopus (104) Google Scholar However, the contribution of aberrantly expressed miRNAs to hepatic cell responses in ALD is unknown. The regulation of miR-34a by the transcription factor p53 suggests a potential role for miR-34 in the modulation of hepatic cell behavior.9He L. He X. Lim L.P. de Stanchina E. Xuan Z. Liang Y. Xue W. Zender L. Magnus J. Ridzon D. Jackson A.L. Linsley P.S. Chen C. Lowe S.W. Cleary M.A. Hannon G.J. A microRNA component of the p53 tumour suppressor network.Nature. 2007; 447: 1130-1134Crossref PubMed Scopus (2260) Google Scholar, 10Chang T.C. Wentzel E.A. Kent O.A. Ramachandran K. Mullendore M. Lee K.H. Feldmann G. Yamakuchi M. Ferlito M. Lowenstein C.J. Arking D.E. Beer M.A. Maitra A. Mendell J.T. Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis.Mol Cell. 2007; 26: 745-752Abstract Full Text Full Text PDF PubMed Scopus (1683) Google Scholar, 11Tarasov V. Jung P. Verdoodt B. Lodygin D. Epanchintsev A. Menssen A. Meister G. Hermeking H. Differential regulation of microRNAs by p53 revealed by massively parallel sequencing: miR-34a is a p53 target that induces apoptosis and G1-arrest.Cell Cycle. 2007; 6: 1586-1593Crossref PubMed Scopus (793) Google Scholar, 12Raver-Shapira N. Marciano E. Meiri E. Spector Y. Rosenfeld N. Moskovits N. Bentwich Z. Oren M. Transcriptional activation of miR-34a contributes to p53-mediated apoptosis.Mol Cell. 2007; 26: 731-743Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar Normally, p53 inhibits cell proliferation and stimulates cell death. However, disruption of the p53 pathway promotes liver injury. One pathway by which p53 regulates cell growth is through miRNA. Cellular stress stabilizes p53 that in turn regulates the expression of a set of miRNA, which control apoptosis and senescence.9He L. He X. Lim L.P. de Stanchina E. Xuan Z. Liang Y. Xue W. Zender L. Magnus J. Ridzon D. Jackson A.L. Linsley P.S. Chen C. Lowe S.W. Cleary M.A. Hannon G.J. A microRNA component of the p53 tumour suppressor network.Nature. 2007; 447: 1130-1134Crossref PubMed Scopus (2260) Google Scholar, 10Chang T.C. Wentzel E.A. Kent O.A. Ramachandran K. Mullendore M. Lee K.H. Feldmann G. Yamakuchi M. Ferlito M. Lowenstein C.J. Arking D.E. Beer M.A. Maitra A. Mendell J.T. Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis.Mol Cell. 2007; 26: 745-752Abstract Full Text Full Text PDF PubMed Scopus (1683) Google Scholar, 11Tarasov V. Jung P. Verdoodt B. Lodygin D. Epanchintsev A. Menssen A. Meister G. Hermeking H. Differential regulation of microRNAs by p53 revealed by massively parallel sequencing: miR-34a is a p53 target that induces apoptosis and G1-arrest.Cell Cycle. 2007; 6: 1586-1593Crossref PubMed Scopus (793) Google Scholar, 12Raver-Shapira N. Marciano E. Meiri E. Spector Y. Rosenfeld N. Moskovits N. Bentwich Z. Oren M. Transcriptional activation of miR-34a contributes to p53-mediated apoptosis.Mol Cell. 2007; 26: 731-743Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar, 13Tazawa H. Tsuchiya N. Izumiya M. Nakagama H. Tumor-suppressive miR-34a induces senescence-like growth arrest through modulation of the E2F pathway in human colon cancer cells.Proc Natl Acad Sci U S A. 2007; 104: 15472-15477Crossref PubMed Scopus (828) Google Scholar Recent studies show that the miRNA miR-34a is activated by p53.9He L. He X. Lim L.P. de Stanchina E. Xuan Z. Liang Y. Xue W. Zender L. Magnus J. Ridzon D. Jackson A.L. Linsley P.S. Chen C. Lowe S.W. Cleary M.A. Hannon G.J. A microRNA component of the p53 tumour suppressor network.Nature. 2007; 447: 1130-1134Crossref PubMed Scopus (2260) Google Scholar, 10Chang T.C. Wentzel E.A. Kent O.A. Ramachandran K. Mullendore M. Lee K.H. Feldmann G. Yamakuchi M. Ferlito M. Lowenstein C.J. Arking D.E. Beer M.A. Maitra A. Mendell J.T. Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis.Mol Cell. 2007; 26: 745-752Abstract Full Text Full Text PDF PubMed Scopus (1683) Google Scholar, 11Tarasov V. Jung P. Verdoodt B. Lodygin D. Epanchintsev A. 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Tumor-suppressive miR-34a induces senescence-like growth arrest through modulation of the E2F pathway in human colon cancer cells.Proc Natl Acad Sci U S A. 2007; 104: 15472-15477Crossref PubMed Scopus (828) Google Scholar, 14Welch C. Chen Y. Stallings R.L. MicroRNA-34a functions as a potential tumor suppressor by inducing apoptosis in neuroblastoma cells.Oncogene. 2007; 26: 5017-5022Crossref PubMed Scopus (692) Google Scholar Although derepression of E2F3 may promote neoplastic growth in tumors in which miR-34a is reduced, such as gliomas, neuroblastomas,14Welch C. Chen Y. Stallings R.L. MicroRNA-34a functions as a potential tumor suppressor by inducing apoptosis in neuroblastoma cells.Oncogene. 2007; 26: 5017-5022Crossref PubMed Scopus (692) Google Scholar and colorectal cancers,13Tazawa H. Tsuchiya N. Izumiya M. Nakagama H. Tumor-suppressive miR-34a induces senescence-like growth arrest through modulation of the E2F pathway in human colon cancer cells.Proc Natl Acad Sci U S A. 2007; 104: 15472-15477Crossref PubMed Scopus (828) Google Scholar the overexpression of miR-34a during human liver regeneration suggests the presence of additional mechanisms by which miR-34a contributes to hepatic cell survival and regeneration.15Chen H. Sun Y. Dong R. Yang S. Pan C. Xiang D. Miao M. Jiao B. Mir-34a is upregulated during liver regeneration in rats and is associated with the suppression of hepatocyte proliferation.PLoS One. 2011; 6: e20238Crossref PubMed Scopus (75) Google Scholar Thus, we assessed the role of aberrant expression of miR-34a in hepatic cell survival and remodeling during ALD by posing the following questions: i) Is miR-34a expression altered in ethanol-exposed mice and ALD human liver tissues? ii) Does modulation of miR-34a alter cell survival and remodeling? iii) Is miR-34a expression be epigenetically modulated? and iv) What downstream targets of miR-34a are involved in ALD? Normal human hepatocytes (N-Heps) and cholangiocytes [human intrahepatic biliary epithelial cells (HiBECs)] were obtained from Sciencell (San Diego, CA). The human hepatocellular cancer cell line, HepG2, was obtained from the American Type Culture Collection (Manassas, VA) and cultured as recommended by the supplier. Cells were grown to ∼75% confluency on 100-mm culture dishes. For studies on the effects of methylation inhibition, cells were incubated with either 10 μmol/L 5-aza-2′deoxycytidine (5-Aza-CdR) or diluent (acetic acid) for 24 hours at 37°C, after which cells were washed twice with cold 1× PBS and harvested for the isolation of genomic DNA or total protein. Six pairs of ALD human liver samples (adjacent liver tissues of HCC patients with heavy alcohol consumption history) and normal control tissues from surgical resections (distal normal liver tissue of patients with liver hemangioma) were analyzed in a masked manner from the Department of Hepatobiliary Surgery, Shengjing Hospital (see Supplemental Table S1 at http://ajp.amjpathol.org). The study protocol of human subjects for the collection of all human materials and data were approved by the Ethics Committee of Shengjing Hospital, China Medical University, Shenyang, China. Transfections were performed by nuclear electroporation using the Nucleofector system (Amaxa Biosystems, Koln, Germany). A total of 50 μL of 100 nmol/L microRNA precursor, antisense inhibitor, or controls (Ambion, Austin, TX) were added to 1 × 106 cells suspended in 50 μL of Nucleofector solution at room temperature. The sequences of the microRNA precursors and inhibitors used can be obtained from Ambion. After electroporation, transfected cells were resuspended in culture medium containing 10% fetal bovine serum for 48 to 72 hours before study. All studies were performed in quadruplicate unless otherwise specified. For long-term alcohol administration, C57BL/6 mice (10 weeks old) were aseptically implanted with gastrostomy catheters as previously described.16Tsukamoto H. Mkrtchyan H. Dynnyk A. Intragastric ethanol infusion model in rodents.Methods Mol Biol. 2008; 447: 33-48Crossref PubMed Scopus (45) Google Scholar, 17Xiong S. She H. Zhang A.S. Wang J. Mkrtchyan H. Dynnyk A. Gordeuk V.R. French S.W. Enns C.A. Tsukamoto H. Hepatic macrophage iron aggravates experimental alcoholic steatohepatitis.Am J Physiol Gastrointest Liver Physiol. 2008; 295: G512-G521Crossref PubMed Scopus (35) Google Scholar An increasing dose of ethanol (22.7 to 35 g/kg per day) or control solutions was infused for 4 weeks. All animal experiments were performed with age- and sex-matched mice from the same littermates and conducted in accordance with the approved Institutional Animal Care and Use Committee protocol at the University of Southern California. RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA). Total RNA (5 μg) was reverse transcribed using biotin end-labeled random octamer oligonucleotide primers. Hybridization of biotin-labeled complementary DNA was performed using a custom miRNA microarray chip (Noncoding RNA Program at Center for Targeted Therapy, M.D. Anderson Cancer Center, Houston, TX), containing 627 probes for mature miRNA corresponding to 324 different human miRNAs spotted in quadruplicate. The images were scanned quantitated using an Axon 4000B scanner (Molecular Devices, Sunnyvale, CA). The scanned images were quantified using GenePix software version 6.0 (Molecular Devices). The data from three samples for each cell type were further analyzed by the BRB-ArrayTools software version 4.2.1 from the National Cancer Institute (Bethesda, MD). The expression of mature miRNAs in human hepatobiliary cell lines was analyzed by TaqMan miRNA Assay (Applied Biosystems, Foster City, CA). Briefly, single-stranded cDNA was synthesized from 10 ng of total RNA in a 15-μL reaction volume by using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). The reactions were incubated first at 16°C for 30 minutes and then at 42°C for 30 minutes. The reactions were inactivated by incubation at 85°C for 5 minutes. Each cDNA generated was amplified by quantitative PCR by using sequence-specific primers from the TaqMan microRNA Assays on a MX 3000P PCR Instrument (Stratagene, San Diego, CA). The 20-μL PCR included 10 μL of 2× Universal PCR Master Mix (No AmpErase UNG), 2 μL of each 10× TaqMan MicroRNA Assay Mix, and 1.5 μL of reverse transcription product. The reactions were incubated in a 96-well plate at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The CT is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. Oligonucleotides were designed to the 5′-promoter region of has-miR-34a genomic sequence, and primers were obtained from Invitrogen. A fragment of 179 to 385 that contains the identified CpG island enriched elements of the 5′- promoter region of has-miR-34a was amplified by PCR and cloned into the MluI and BgIII sites of the pGL3-luciferase plasmid (Promega, Madison, WI) to form 5′-miR-34a-LUC; pCMV-Renilla was obtained from Promega. A 24-bp mutation of the hsa-miR-34a methylation site (206 bp) GCC to TAA was performed using oligonucleotides to the CpG island enriched region and a Quikchange site mutagenesis kit (Stratagene, San Diego, CA) according to the manufacturer's instructions to form miR-34a-MUT. Orientation and sequence identity were confirmed in all plasmids by sequencing. RNA was isolated from colon tissues using TRIzol (Invitrogen) and cleaned with the Qiagen's RNeasy Kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. The optional on-column DNase treatment was performed. Reverse transcription was performed using 1 μg of RNA with SABios-ciences' RT2 First Strand Kit (SABiosciences, Frederick, MD) according to the manufacturer's protocol. Mouse liver tissue cDNA was analyzed using SuperArray plates (Epigenetic Chromatin Modification Enzymes PCR Array, SABiosciences). To validate the translational significance of these gene expression findings, mice liver samples were analyzed using real-time PCR. RT2 qPCR Primer Assays were obtained from SABiosciences. Real-time PCR was performed using SABiosciences RT2 SYBR Green/ROX qPCR Master Mix for a Stratagene Mx3005P Real-Time PCR System according to the manufacturer protocol. ROX was used as an endogenous reference, and data were analyzed using SABiosciences PCR Array Data Analysis Template. The comparative CT method (ΔΔCT) was used for quantification of gene expression. All samples were tested in triplicate and mean values used for quantification. The mRNA levels of DNMT3B and DNMT1 were analyzed by semiquantitative RT-PCR. A total of 1 μg of total RNA, isolated using an RNA isolation kit from Invitrogen, was used in reverse transcription reactions as described by the manufacturer. The resulting total cDNA was used in the PCR reaction to measure the mRNA levels of DNMT3B and DNMT1. The mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal control. PCR was performed with Taq polymerase, and conditions were as follows: predenaturing at 94°C for 3 minutes and 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 60 seconds. The PCR cycle numbers were 26 for GAPDH, 30 for DNMT1, and 35 for DNMT3B. The primers used for RT-PCR were as follows: DNMT3B: sense primer, 5′-AATGTGAATCCAGCCAGGAAAGGC-3′; antisense primer, 5′-ACTGGATTACACTCCAGGAACCGT-3′; DNMT1: sense primer, 5′-ACCGCTTCTACTTCCTCGAGGCCTA-3′; antisense primer, 5′-GTTGCAGTCCTCTGTGAACACTGTGG-3′; GAPDH: sense primer, 5′-AAGGCTGAGAACGGGAAGCTTGTCATCAAT-3′; antisense primer, 5′-TTCCCGTTCAGCTCAGGGATGACCTTGCCC-3′. Under the conditions used, the cDNAs were exponentially amplified, and thus a semiquantitative estimation of the products was possible. The cell lysate samples (500 μg) were reduced, alkylated, and double digested with trypsin to generate peptides. The digested peptides were dried in a SpeedVac and resuspended in 100 μL of 0.1% formic acid in 5% acetonitrile (mobile phase A). A total of 200 μg of peptides (40 μL) was directly loaded onto a 1 × 150-mm Poly-SEA strong cation exchange column (Michrom Bioresources, Auburn, CA) using an Agilent 1200 autosampler (Agilent Technologies, Santa Clara, CA). Peptides were eluted to 10 fractions using 0 to 100 mmol/L ammonium formate for 40 minutes (mobile phase B: 1 mmol/L ammonium formate, 10% formic acid in 5% acetonitrile) and five fractions in 100 to 1000 mmol/L ammonium formate for 10 minutes (on an Agilent 1200 Capillary LC and Analytical-Fraction Collector at a flow rate of 50 μL/min). Peptides were completely dried and reconstituted in 20 μL of 0.1% trifluoroacetic acid for liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis using an LC/MS system consisting of an 1100 Series liquid chromatograph, HPLC-Chip Cube MS interface, and 1100 Series LC/MSD Trap XCT Ultra ion trap mass spectrometer (all Agilent Technologies). The system was equipped with an HPLC-Chip (Agilent Technologies) that incorporated a 40-nL enrichment column and a 43 × 75-μm analytical column packed with Zorbax 300SB-C18 5-μm particles. The gain control (intraclass correlation coefficient) was set to 500,000 with a maximum accumulation time of 150 milliseconds. The coefficient of intrinsic dependence was triggered on the six most abundant, not singly charged peptide ions in the m/z range of 450 to 1500. Precursors were set in an exclusion list for 1 minute after two MS/MS spectra. Coefficient of intrinsic dependence data were searched against the NCBInr human database, using the Agilent Spectrum Mill Server software version Rev A.03.03 (Agilent Technologies). A Spectrum Mill autovalidation was performed first in the protein details followed by peptide mode using default values [minimum scores, minimum scored peak intensity, forward minus reversed score threshold, and rank 1 minus rank 2 score threshold]. All protein hits found in a distinct database search by Spectrum Mill are nonredundant. Cells grown in 100-mm dishes were lysed and protein content quantitated using the Bradford protein assay. Equivalent amounts of protein were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked and incubated with the specific primary antibody overnight at 4°C, washed, and incubated with the appropriate IRDye700- and IRDye800-labeled secondary antibodies (Rockland, Gilbertsville, PA) (1:1000) for 1 hour. Blots were stripped and reprobed with mouse monoclonal antibodies for α-tubulin (Sigma Chemical, St. Louis, MO) (1:2000), which was used for normalization. Protein expression was visualized and quantified using the LI-COR Odyssey Infrared Imaging System (LI-COR Bioscience, Lincoln, NE). Real-time PCR assays were performed as described.18Francis H. Franchitto A. Ueno Y. Glaser S. DeMorrow S. Venter J. Gaudio E. Alvaro D. Fava G. Marzioni M. Vaculin B. Alpini G. H3 histamine receptor agonist inhibits biliary growth of BDL rats by downregulation of the cAMP-dependent PKA/ERK1/2/ELK-1 pathway.Lab Invest. 2007; 87: 473-487PubMed Google Scholar, 19Alpini G. Franchitto A. Demorrow S. Onori P. Gaudio E. Wise C. Francis H. Venter J. Kopriva S. Mancinelli R. Carpino G. Stagnitti F. Ueno Y. Han Y. Meng F. Glaser S. Activation of alpha(1) -adrenergic receptors stimulate the growth of small mouse cholangiocytes via calcium-dependent activation of nuclear factor of activated T cells 2 and specificity protein 1.Hepatology. 2011; 53: 628-639Crossref PubMed Scopus (28) Google Scholar Briefly, cDNA was generated by reverse transcription using 1 μg of total RNA isolated using the RNA isolation kit (Bio-Rad, Hercules, CA) and the SuperScript III Reverse Transcription Kit (Invitrogen). Quantitative real-time PCR was performed on a MX 3005P PCR Instrument (Stratagene, San Diego, CA) as described.18Francis H. Franchitto A. Ueno Y. Glaser S. DeMorrow S. Venter J. Gaudio E. Alvaro D. Fava G. Marzioni M. Vaculin B. Alpini G. H3 histamine receptor agonist inhibits biliary growth of BDL rats by downregulation of the cAMP-dependent PKA/ERK1/2/ELK-1 pathway.Lab Invest. 2007; 87: 473-487PubMed Google Scholar, 19Alpini G. Franchitto A. Demorrow S. Onori P. Gaudio E. Wise C. Francis H. Venter J. Kopriva S. Mancinelli R. Carpino G. Stagnitti F. Ueno Y. Han Y. Meng F. Glaser S. Activation of alpha(1) -adrenergic receptors stimulate the growth of small mouse cholangiocytes via calcium-dependent activation of nuclear factor of activated T cells 2 and specificity protein 1.Hepatology. 2011; 53: 628-639Crossref PubMed Scopus (28) Google Scholar Each sample was tested in triplicate. The PCR products were verified by melting curve analysis and by 1.8% agarose gel electrophoresis of the PCR product. Matrix metalloproteinase (MMP) mRNA expression was normalized against expression of β-actin used as an internal control. N-Heps, HiBECs, and HepG2 cells (5 × 104 cells) were placed into the top chamber of a BD Falcon HTS FluoroBlok insert with a membrane containing 8-μm pores (BD Biosciences, Rockville, MD) in 300 μL of serum-free Dulbecco's modified Eagle medium in triplicate. The inserts were placed into the bottom chamber wells of a 96-well plate containing Dulbecco's modified Eagle medium media and fetal bovine serum (5%) as a chemoattractant. Cells that migrated through the pores of the membrane to the bottom chamber were labeled with 8 μg/mL of calcein-AM (Molecular Probes, Eugene, OR) in PBS for 30 minutes at 37°C. The fluorescence of migrated cells was quantified using a fluorometer at excitation wavelengths of 485 nm and emission wavelengths of 530 nm and expressed as arbitrary fluorescence units. Cell proliferation was measured using the CellTiter 96 AQueous Assay Kit (Promega). Transfected cells (10,000 per well) were plated in 96-well plates (BD Biosciences) and incubated at 37°C, and cell proliferation was assessed after 72 hours as previously described.5Meng F. Glaser S.S. Francis H. Demorrow S. Han Y. Passarini J.D. Stokes A. Cleary J.P. Liu X. Venter J. Kumar P. Priester S. Hubble L. Stoloch D. Sharma J. Liu C.G. Alpini G. Functional analysis of microRNAs in human hepatocellular cancer stem cells.J Cell Mol Med. 2012; 16: 160-173Crossref PubMed Scopus (104) Google Scholar, 19Alpini G. Franchitto A. Demorrow S. Onori P. Gaudio E. Wise C. Francis H. Venter J. Kopriva S. Mancinelli R. Carpino G. Stagnitti F. Ueno Y. Han Y. Meng F. Glaser S. Activation of alpha(1) -adrenergic receptors stimulate the growth of small mouse cholangiocytes via calcium-dependent activation of nuclear factor of activated T cells 2 and specificity protein 1.Hepatology. 2011; 53: 628-639Crossref PubMed Scopus (28) Google Scholar N-Heps, HiBECs, and HepG2 cells were seeded in 96-well plates (10,000 per well) in modified Dulbecco's modified Eagle medium with 10% fetal bovine serum after miRNA transfection. The final concentration of the bottom and top feeder layers of the agar system was 0.6%, and the cell suspension layer was 0.4%. Anchorage-independent transformed cell growth was fluorometrically assayed after 7 days using Alamar Blue (Biosource International, Camarillo, CA), and the SpectraMax M5 Multi-Mode Microplate Reader (Molecular Devices Inc, Sunnyvale, CA; excitation, 530/25 nm; emi
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