MicroRNA-26a Targets the Histone Methyltransferase Enhancer of Zeste homolog 2 during Myogenesis
2008; Elsevier BV; Volume: 283; Issue: 15 Linguagem: Inglês
10.1074/jbc.m709614200
ISSN1083-351X
AutoresChung F. Wong, Ross L. Tellam,
Tópico(s)Circular RNAs in diseases
ResumoMicroRNA (miRNA) are important regulators of many bio×logical processes, but the targets for most miRNA are still poorly defined. In this study, we profiled the expression of miRNA during myogenesis, from proliferating myoblasts through to terminally differentiated myotubes. Microarray results identified six significantly differentially expressed miRNA that were more than 2-fold different in myotubes. From this list, miRNA-26a (miR-26a), an up-regulated miRNA, was further examined. Overexpression of miR-26a in murine myogenic C2C12 cells induced creatine kinase activity, an enzyme that markedly increases during myogenesis. Further, myoD and myogenin mRNA expression levels were also up-regulated. These results suggest that increased expression of miR-26a promotes myogenesis. Through a bioinformatics approach, we identified the histone methyltransferase, Enhancer of Zeste homolog 2 (Ezh2), as a potential target of miR-26a. Overexpression of miR-26a suppressed the activity of a luciferase reporter construct fused with the 3′-untranslated region of Ezh2. In addition, miR-26a overexpression decreased Ezh2 mRNA expression. These results reveal a model of regulation during myogenesis whereby the up-regulation of miR-26a acts to post-transcriptionally repress Ezh2, a known suppressor of skeletal muscle cell differentiation. MicroRNA (miRNA) are important regulators of many bio×logical processes, but the targets for most miRNA are still poorly defined. In this study, we profiled the expression of miRNA during myogenesis, from proliferating myoblasts through to terminally differentiated myotubes. Microarray results identified six significantly differentially expressed miRNA that were more than 2-fold different in myotubes. From this list, miRNA-26a (miR-26a), an up-regulated miRNA, was further examined. Overexpression of miR-26a in murine myogenic C2C12 cells induced creatine kinase activity, an enzyme that markedly increases during myogenesis. Further, myoD and myogenin mRNA expression levels were also up-regulated. These results suggest that increased expression of miR-26a promotes myogenesis. Through a bioinformatics approach, we identified the histone methyltransferase, Enhancer of Zeste homolog 2 (Ezh2), as a potential target of miR-26a. Overexpression of miR-26a suppressed the activity of a luciferase reporter construct fused with the 3′-untranslated region of Ezh2. In addition, miR-26a overexpression decreased Ezh2 mRNA expression. These results reveal a model of regulation during myogenesis whereby the up-regulation of miR-26a acts to post-transcriptionally repress Ezh2, a known suppressor of skeletal muscle cell differentiation. MicroRNA (miRNA) 2The abbreviations used are:miRNAmicroRNAUTRuntranslated regionRT-PCRreal-time PCRqRT-PCRquantitative RT PCRMes4-morpholineethanesulfonic acidDAPI4′,6-diamidino-2-phenylindole. are a class of small, non-coding RNA important in post-transcriptional gene silencing. They are thought to target the expression of approximately one-third of all mammalian genes (1Kim V.N. Nat. Rev. Mol. Cell. Biol. 2005; 6: 376-385Crossref PubMed Scopus (1999) Google Scholar, 2Lewis B.P. Burge C.B. Bartel D.P. Cell. 2005; 120: 15-20Abstract Full Text Full Text PDF PubMed Scopus (9882) Google Scholar). miRNA are derived from endogenous transcribed RNA hairpin structures found within introns, exons, or intergenic regions of the genome (3Lagos-Quintana M. Rauhut R. Meyer J. Borkhardt A. Tuschl T. RNA (Cold Spring Harbor). 2003; 9: 175-179Google Scholar). The biogenesis of the 19–24-nucleotide mature miRNA involves a two-step cleavage process by two ribonuclease III enzymes (4Lee Y. Jeon K. 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Subsequent studies demonstrated that miR-1 overexpression in HeLa cells caused the direct or indirect down-regulation of more than 100 mRNA transcripts and altered the global expression profile of these cells to resemble that of muscle (32Lim L.P. Lau N.C. Garrett-Engele P. Grimson A. Schelter J.M. Castle J. Bartel D.P. Linsley P.S. Johnson J.M. Nature. 2005; 433: 769-773Crossref PubMed Scopus (4007) Google Scholar). Interestingly, although miR-1 Drosophila knock-out larvae developed normal musculature, they died as small second instar larvae due to severe muscular deformity, indicating that miR-1 is not involved in muscle formation but rather in the maintenance of differentiation (31Sokol N.S. Ambros V. Genes Dev. 2005; 19: 2343-2354Crossref PubMed Scopus (344) Google Scholar). In the mouse myoblast cell line C2C12, miR-1, miR-133a, and miR-206 promote myoblast differentiation (15Kim H.K. Lee Y.S. Sivaprasad U. Malhotra A. Dutta A. J. Cell Biol. 2006; 174: 677-687Crossref PubMed Scopus (646) Google Scholar, 27Chen J.F. Mandel E.M. Thomson J.M. Wu Q. Callis T.E. Hammond S.M. Conlon F.L. Wang D.Z. Nat. Genet. 2006; 38: 228-233Crossref PubMed Scopus (2238) Google Scholar). Recently, it was also demonstrated that Dicer plays an essential role in skeletal muscle development (33O'Rourke J.R. Georges S.A. Seay H.R. Tapscott S.J. McManus M.T. Goldhamer D.J. Swanson M.S. Harfe B.D. Dev. Biol. 2007; 311: 359-368Crossref PubMed Scopus (270) Google Scholar). These results suggest that miRNA regulate myogenesis at various stages by silencing genes that repress the differentiation process. To further investigate this hypothesis, we profiled miRNA expression during myogenesis of the mouse myoblast cell line, C2C12. These cells have been extensively studied and differentiate into myotubes under specific culture conditions (34Yaffe D. Saxel O. Nature. 1977; 270: 725-727Crossref PubMed Scopus (1563) Google Scholar). From the identified differentially expressed miRNA, we further examined the role of miR-26a in regulating myogenesis. Using a specific bioinformatics approach, we predicted and then experimentally tested a potential target of miR-26a, Enhancer of Zeste homolog 2 (Ezh2), a negative regulator of myogenesis. Cell Culture—The C2C12 mouse myoblast cell line, first derived from adult mouse dystrophic muscle (34Yaffe D. Saxel O. Nature. 1977; 270: 725-727Crossref PubMed Scopus (1563) Google Scholar), was acquired from the American Type Culture Collection (ATCC). Myoblasts were maintained in growth medium consisting of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 10 units/ml penicillin/streptomycin/glutamine. Once confluent, the medium was changed to Dulbecco's modified Eagle's medium supplemented with 2% horse serum and 10 units/ml penicillin/streptomycin/glutamine to promote myoblast differentiation into myocytes and subsequent fusion into multinucleated myotubes. Immunoblotting and Immunofluorescence Localizations—Total cellular protein (30 μg) was subjected to SDS-PAGE electrophoresis and standard immunoblotting techniques (35Khanna K.K. Beamish H. Yan J. Hobson K. Williams R. Dunn I. Lavin M.F. Oncogene. 1995; 11: 609-618PubMed Google Scholar). The following primary antibodies and titers were used: anti-desmin (antibody D1033, Sigma-Aldrich, 1/5000) and anti-Ezh2 (antibody 4905, Cell Signaling, 1/500). Immunoreactive bands were detected using species-specific horseradish peroxidase-conjugated secondary antibodies (Chemicon, 1/3000) and visualized using the Supersignal West Pico chemiluminescent substrate (Pierce, Quantum Scientific). For immunofluorescence localization studies, C2C12 cells grown on coverslips were fixed in cold methanol at various differentiation time points (36Wong C.F. Barnes L.M. Smith L. Popa C. Serewko-Auret M.M. Saunders N.A. Biochem. Biophys. Res. Commun. 2004; 324: 497-503Crossref PubMed Scopus (11) Google Scholar). Immunolocalizations used antibodies against desmin (Sigma-Aldrich, diluted 1/100) and Ezh2 (catalog number 36-6300, Invitrogen, diluted 1/50). Desmin localization was visualized using an anti-mouse fluorescein isothiocyanate-conjugated secondary antibody (AQ326F, Chemicon, diluted, 1/150), and Ezh2 localization was visualized using an anti-rabbit AlexaFluor 555-conjugated antibody (A21429, Invitrogen, diluted 1/100). The nuclei of the cells were visualized using DAPI staining. Creatine Kinase Activity Assay—C2C12 cells were harvested in 50 mm Tris-Mes buffer (pH 7.8) containing 1% v/v Triton X-100 and stored at -80 °C. To measure creatine kinase enzyme activity, 100 μl of N-acetyl cysteine (TR14010, Thermo Electron Corp.) was added to 5 μl of cell extracts and absorbance read at 340 nm every 20 s over 5 min. The rate of reaction over this period was linear and used as the measure of enzyme activity normalized to total protein concentration (i.e. specific enzyme activity; units/mg). Isolation of Total RNA—Total RNA was isolated from C2C12 cells at the following time points: proliferating (70–80% confluence), confluent (100% confluence), 1 day (+1d), 2 days (+2d), and 4 days (+4d) (see Figs. 1 and 4) after induction of differentiation. Total RNA was isolated using TRIzol (Invitrogen) as per the manufacturer's instructions. The purity and concentration of the RNA was determined by measuring its absorbance at 260 and 280 nm.FIGURE 4Ezh2 is a target for miR-26a in C2C12 cells. a, expression of Ezh2 mRNA during myogenesis, as measured by qRT-PCR. MNE, mean normalized expression; prol, proliferating; conf, confluent; +1d, 1 day; +2d, 2 days; +4d, 4 days after induction of differentiation. b, protein expression of Ezh2 during myogenesis, as assayed by immunoblotting. c, co-localization of Ezh2 (red) with DAPI (blue) in the nucleus of C2C12 myoblasts (proliferating (prol)) and myotubes (+4d). d, C2C12 myoblasts were transfected with a luciferase reporter gene linked to the 3′-UTR of Ezh2 (Ezh2-Luc) or Ezh2 3′-UTR with a mutated miR-26a recognition site (Ezh2mut-Luc) and co-transfected with miR-26a or a scrambled control expression plasmid. Each measurement was made in triplicate. A.U., arbitrary units. e and f, C2C12 myoblasts were transiently transfected with miR-26a or a scrambled control expression plasmid. Total RNA and whole cell extracts were harvested 48 h after transfection and assayed for Ezh2 mRNA expression by qRT-PCR (e) or protein expression by immunoblotting (f). Data presented as mean ± S.E. of three independent experiments. Each measurement was made in triplicate. ***, p < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) miRNA Microarray—The miRCURY™ locked-nucleic acid array version 8.1 (Exiqon) consists of probes for the mature forms of all miRNA present in the miRBase 8.1 release of the miRNA registry (37Griffiths-Jones S. Grocock R.J. van Dongen S. Bateman A. Enright A.J. Nucleic Acids Res. 2006; 34: D140-D144Crossref PubMed Scopus (3656) Google Scholar, 38Griffiths-Jones S. Nucleic Acids Res. 2004; 32: D109-D111Crossref PubMed Google Scholar). The microarray contains probes for 1488 mature miRNA, each represented twice on the microarray. Since the mature sequence of an miRNA is highly conserved between species, it is possible that cross-species hybridization could occur. However, the locked-nucleic acid miRNA probes are highly sensitive and optimized to minimize cross-hybridization between similar mature miRNA (39Castoldi M. Schmidt S. Benes V. Noerholm M. Kulozik A.E. Hentze M.W. Muckenthaler M.U. RNA (Cold Spring Harbor). 2006; 12: 913-920Google Scholar). The Adelaide Microarray Facility (University of Adelaide, South Australia) printed the microarray slides and hybridized the samples. The reference RNA (see Fig. 1, reference) consisted of a pool of total RNA isolated from four independent proliferating C2C12 samples. Each time point was represented by four biological replicates, of which two were labeled with Cy3 and two were labeled with Cy5 according to a standard protocol (39Castoldi M. Schmidt S. Benes V. Noerholm M. Kulozik A.E. Hentze M.W. Muckenthaler M.U. RNA (Cold Spring Harbor). 2006; 12: 913-920Google Scholar). For each sample, 1 μg of total RNA was labeled and hybridized to the microarray. Microarrays were scanned on a ScanArray 4000 XL scanner (Packard BioChip Technologies), and raw data were extracted using Spot software (40Wilson D.L. Buckley M.J. Helliwell C.A. Wilson I.W. Bioinformatics (Oxf.). 2003; 19: 1325-1332Crossref PubMed Scopus (76) Google Scholar). Data were subsequently analyzed using GeneSpring® version 7.2 (Agilent Technologies), and statistical analysis was performed using background-corrected mean signal intensities from each dye channel. Microarray data were normalized using intensity-dependent global normalization (Lowess). Differentially expressed miRNA were identified using one-way analysis of variance (p < 0.05) and the Benjamini and Hochberg false discovery rate with Tukey's honestly significant differences post hoc test to minimize selection of false positives. Of the significantly differentially expressed miRNA, only those with greater than 2-fold increase or 2-fold decrease in expression at any time point when compared with the proliferating sample were used for further analysis. This conservative approach focused on significantly differentially expressed miRNA that showed the greatest changes in expression during myogenesis. All microarray data presented in this manuscript are in accordance with Minimum Information About a Microarray Experiment (MIAME) guidelines and have been deposited in the National Center for Biotechnology Information (NCBI) GEO data base (accession number: GSE9449). To further analyze differentially expressed miRNA, hierarchical gene-tree clustering and K-means clustering analysis, both using default parameters, were used to group miRNA with similar expression profiles (41Hartigan J. Wang M. Appl. Stat. 1979; 28: 100-108Crossref Google Scholar). Taqman® miRNA Expression Assays—RNA was reverse-transcribed using specific miRNA stem-loop primers (42Chen C. Ridzon D.A. Broomer A.J. Zhou Z. Lee D.H. Nguyen J.T. Barbisin M. Xu N.L. Mahuvakar V.R. Andersen M.R. Lao K.Q. Livak K.J. Guegler K.J. Nucleic Acids Res. 2005; 33: e179Crossref PubMed Scopus (4062) Google Scholar) and the Taqman® miRNA reverse transcription kit (Applied Biosystems). Mature miRNA expression was measured with Taqman® microRNA assays (Applied Biosystems) according to the manufacturer's instructions. The expression of the miRNA was normalized against the expression level of the house-keeping gene, RpPO (NM_007475) or GAPDH (NM_008084), and presented as the mean normalized expression. Construction of Expression Plasmids—The following primer pair was used to clone the mouse miR-26a precursor into the pSilencer™ 4.1-CMV vector (Ambion, Geneworks) according to the manufacturer's instructions: miR-26a 5′-GGATCCGCAGAAACTCCAGAGAGAAGGA-3′;3′-AAGCTTGCCTTTAGCAGAAAGGAGGTT-5′. Primers contained 5′-BamHI and 3′-HindIII restriction sites (underlined) to facilitate cloning into the vector. The following primers were used to amplify and clone the 3′-UTR of mouse Ezh2 into the pMIR-REPORT™ luciferase vector (Ambion, Geneworks) according to manufacturer's instructions: 5′-ATACGCGTCTTGACATCTACTACCTCTTC-3′; 3′-ATAAGCTTACAAGTTCAAGTATTCTTTA-5′. The forward primer includes a MluI restriction site, and the reverse primer has a HindIII site (underlined) to facilitate ligation into the vector. The predicted miR-26a binding site (TACTTGAA, italicized) located in the 3′-UTR of Ezh2 was mutated by a single base pair change (underlined) using DpnI mediated site-directed mutagenesis (43Fisher C.L. Pei G.K. BioTechniques. 1997; 23: 570-574Crossref PubMed Scopus (292) Google Scholar). Primers used for this procedure were as follows: 5′-GAATAAAGAATGCTTGAACTTG-3′;3′-CAAGTTCAAGCATTCTTTATTC-5′. The inserts from all cloned plasmids were sequenced by the Australian Genome Research Facility (Queensland, Australia) to verify identities. Transfections—For miRNA overexpression studies, C2C12 myoblasts were seeded at a cell density of 2.0 × 105 cells/well in 6-well plates with OptiMEM® I medium (Invitrogen) 1 day prior to transfection. The following day, cells were transfected with expression plasmids using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's instructions. Each reaction consisted of 5 μg of expression plasmid DNA (pSilencer™ expression plasmid expressing miR-26a or a scrambled siRNA hairpin control with no homology to any known sequences in the human, mouse, or rat genomes) and 12.5 μl of Lipofectamine™ 2000. To visualize the transfection, an expression plasmid coding for red fluorescent protein was co-transfected in a 1:4 ratio to the pSilencer™ expression plasmid. The transfection reagents were removed after 4 h and replaced with growth medium. Cells were harvested for subsequent assays 48 h after transfection. Transfections of C2C12 myoblasts were also performed in a 96-well plate for creatine kinase activity assays using 0.3 μg of pSilencer™ expression plasmid encoding miR-26a or a scrambled control and 0.75 μl of Lipofectamine™ 2000. In addition, C2C12 myoblasts transfected with these plasmids were also induced to differentiate by changing the growth medium to Dulbecco's modified Eagle's medium supplemented with 2% horse serum and 10 units/ml penicillin/streptomycin/glutamine. The cells were then visualized by immunofluorescence staining of desmin. For luciferase reporter assays, 5 × 103 cells were initially plated in a 96-well plate. Constructs were transfected into the C2C12 myoblasts at the following concentrations: 0.3 μg of reporter gene (Ezh2-Luc or Ezh2mut-Luc), 0.1 μg of β-galactosidase control plasmid, and 0.1 μg of pSilencer™ expression plasmid encoding miR-26a or a scrambled control. Transfections were performed in triplicate, and the experiment repeated three times. Twenty-four hours after transfection, luciferase and β-galactosidase activities were measured using the DualLight® assay system (Applied Biosystems). Luciferase activity was normalized for transfection efficiency by measuring β-galactosidase control activity according to the manufacturer's instructions. For miR-26a inhibition assays, 5 × 104 C2C12 myoblasts were plated in a 24-well plate. Cells were transfected with either the miRIDIAN™ microRNA inhibitor designed against miR-26a or the scrambled sequence negative control (C-310118-04 or IN-001000-01, respectively; Dharmacon, Millennium Science). The inhibitor or control were both transfected into cells (250 ng) using Lipofectamine 2000, as per the manufacturer's instructions. Cells were harvested 48 h after transfection and assayed for creatine kinase activity, myogenin, and myoD mRNA expression. Direct transfection of the miRIDIAN™ microRNA inhibitor into C2C12 myotubes was not possible. Statistical significance of results was calculated using the Student's t test, where p < 0.05 was considered significant. Identification of Putative mRNA Targets for miRNA Up-regulated during Myogenesis—mRNA targets for miRNA significantly up-regulated during myogenesis were predicted using the computer programs PicTar (44Krek A. Grun D. Poy M.N. Wolf R. Rosenberg L. Epstein E.J. Macmenamin P. da Piedade I. Gunsalus K.C. Stoffel M. Rajewsky N. Nat. Genet. 2005; 37: 495-500Crossref PubMed Scopus (3903) Google Scholar), miRanda (45John B. Enright A.J. Aravin A. Tuschl T. Sander C. Marks D.S. PLoS Biol. 2004; 2: e363Crossref PubMed Scopus (2955) Google Scholar), and Targetscan (46Lewis B.P. Shih I.H. Jones-Rhoades M.W. Bartel D.P. Burge C.B. Cell. 2003; 115: 787-798Abstract Full Text Full Text PDF PubMed Scopus (4225) Google Scholar). To minimize the number of false predictions, only mRNA targets predicted using all three programs were considered. Predicted mRNA targets of miRNA were then compared with mRNA that were significantly down-regulated during C2C12 differentiation (47Tomczak K.K. Marinescu V.D. Ramoni M.F. Sanoudou D. Montanaro F. Han M. Kunkel L.M. Kohane I.S. Beggs A.H. FASEB J. 2004; 18: 403-405Crossref PubMed Scopus (151) Google Scholar). Quantitative Real-time PCR (qRT-PCR)—The qRT-PCR procedure has been previously described (48Vuocolo T. Byrne K. White J. McWilliam S. Reverter A. Cockett N.E. Tellam R.L. Physiol. Genomics. 2007; 28: 253-272Crossref PubMed Scopus (69) Google Scholar). Expression was normalized to the housekeeping gene, GAPDH, which remained constant throughout the experiment (49Berry F.B. Miura Y. Mihara K. Kaspar P. Sakata N. Hashimoto-Tamaoki T. Tamaoki T. J. Biol. Chem. 2001; 276: 25057-25065Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The primers used for qRT-PCR to quantitate mRNA expression in C2C12 cells were as follows: Ezh2, 5′-GAAAAAGGACGGCTCCTCTAA-3′, 3′-CATGGACACTGTTTGGTGTTG-5′; GAPDH, 5′-CCTGGAGAAACCTGCCAAGT-3′, 3′-AGCCGTATTCATTGTCATACCA-5′; myoD, 5′-CATCCCTAAGCGACACAGAAC-3′, 3′-AGCACCTGATAAATCGCATTG-5′; and myogenin, 5′-GCAGGCTCAAGAAAGTGAATG-3′, 3′-TAGGCGCTCAATGTACTGGAT-5′. Four technical replicates were used for each assay. miRNA Are Differentially Expressed during Myogenesis—Expression profiles of miRNA during C2C12 differentiation were examined using a commercial miRNA microarray that contained all miRNA present in the miRBase miRNA registry release 8.1 (37Griffiths-Jones S. Grocock R.J. van Dongen S. Bateman A. Enright A.J. Nucleic Acids Res. 2006; 34: D140-D144Crossref PubMed Scopus (3656) Google Scholar, 38Griffiths-Jones S. Nucleic Acids Res. 2004; 32: D109-D111Crossref PubMed Google Scholar). Expression patterns of miRNA at confluence, 1, 2, and 4 days after induction of differentiation were compared with proliferating myoblasts (reference myoblasts) (Fig. 1a). Each time point was represented by four independent biological samples. Microarray analysis revealed that six miRNA were significantly differentially expressed by greater than 2-fold during the course of myogenesis (Fig. 1b). Five miRNA (miR-133a/miR-133b, mml-miR-133a (Macaca mulatta miR133a), miR-206, miR-26a, and miR-422b) were up-regulated, and one was down-regulated (miR-222). Using the default K-means clustering analysis in GeneSpring® version 7.2 (Agilent Technologies), the differentially expressed miRNA were grouped into three clusters (Fig. 1c). Cluster I represents a group of three miRNA that were rapidly up-regulated as C2C12 cells committed to myogenesis. This cluster of miRNA contains miR-206, miR-133a/miR-133b, and mml-miR-133a, a shorter form of miR-133a from M. mulatta that differs in sequence by one base. miR-133a and miR-133b are located at different genetic loci and differ in their mature sequences by one nucleotide in their 3′-ends. These miRNA were previously shown to be highly expressed in skeletal muscle (30Sempere L.F. Freemantle S. Pitha-Rowe I. Moss E. Dmitrovsky E. Ambros V. Genome Biol. 2004; 5: R13Crossref PubMed Google Scholar, 50Liu C.G. Calin G.A. Meloon B. Gamliel N. Sevignani C. Ferracin M. Dumitru C.D. Shimizu M. Zupo S. Dono M. Alder H. Bullrich F. Negrini M. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9740-9744Crossref PubMed Scopus (823) Google Scholar). The miRNA in Cluster II (miR-
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