miR-199a*, a Bone Morphogenic Protein 2-responsive MicroRNA, Regulates Chondrogenesis via Direct Targeting to Smad1
2009; Elsevier BV; Volume: 284; Issue: 17 Linguagem: Inglês
10.1074/jbc.m807709200
ISSN1083-351X
AutoresEdward Lin, Li Kong, Xiaohui Bai, Yi Luan, Liu C,
Tópico(s)Circular RNAs in diseases
ResumoMicroRNAs (miRNA) are short non-coding RNA molecules that regulate a variety of biological processes. The role of miRNAs in BMP2-mediated biological processes is of considerable interest. A comparative miRNA array led to the isolation of several BMP2-responsive miRNAs. Among them, miR-199a* is of particular interest, because it was reported to be specifically expressed in the skeletal system. Here we demonstrate that miR-199a* is an early responsive target of BMP2: its level was dramatically reduced at 5 h, quickly increased at 24 h and remained higher thereafter in the course of BMP2-triggered chondrogenesis of a micromass culture of pluripotent C3H10T1/2 stem cells. miR-199a* significantly inhibited early chondrogenesis, as revealed by the reduced expression of early marker genes for chondrogenesis such as cartilage oligomeric matrix protein (COMP), type II collagen, and Sox9, whereas anti-miR-199a* increased the expression of these chondrogenic marker genes. A computer-based prediction algorithm led to the identification of Smad1, a well established downstream molecule of BMP-2 signaling, as a putative target of miR-199a*. The pattern of Smad1 mRNA expression exhibited the mirror opposite of miR-199a* expression following BMP-2 induction. Furthermore, miR-199a* demonstrated remarkable inhibition of both endogenous Smad1 as well as a reporter construct bearing the 3-untranslated region of Smad1 mRNA. In addition, mutation of miR-199a* binding sites in the 3′-untranslated region of Smad1 mRNA abolished miR-199a*-mediated repression of reporter gene activity. Mechanism studies revealed that miR-199a* inhibits Smad1/Smad4-mediated transactivation of target genes, and that overexpression of Smad1 completely corrects miR-199a*-mediated repression of early chondrogenesis. Taken together, miR-199a* is the first BMP2 responsive microRNA found to adversely regulate early chondrocyte differentiation via direct targeting of the Smad1 transcription factor. MicroRNAs (miRNA) are short non-coding RNA molecules that regulate a variety of biological processes. The role of miRNAs in BMP2-mediated biological processes is of considerable interest. A comparative miRNA array led to the isolation of several BMP2-responsive miRNAs. Among them, miR-199a* is of particular interest, because it was reported to be specifically expressed in the skeletal system. Here we demonstrate that miR-199a* is an early responsive target of BMP2: its level was dramatically reduced at 5 h, quickly increased at 24 h and remained higher thereafter in the course of BMP2-triggered chondrogenesis of a micromass culture of pluripotent C3H10T1/2 stem cells. miR-199a* significantly inhibited early chondrogenesis, as revealed by the reduced expression of early marker genes for chondrogenesis such as cartilage oligomeric matrix protein (COMP), type II collagen, and Sox9, whereas anti-miR-199a* increased the expression of these chondrogenic marker genes. A computer-based prediction algorithm led to the identification of Smad1, a well established downstream molecule of BMP-2 signaling, as a putative target of miR-199a*. The pattern of Smad1 mRNA expression exhibited the mirror opposite of miR-199a* expression following BMP-2 induction. Furthermore, miR-199a* demonstrated remarkable inhibition of both endogenous Smad1 as well as a reporter construct bearing the 3-untranslated region of Smad1 mRNA. In addition, mutation of miR-199a* binding sites in the 3′-untranslated region of Smad1 mRNA abolished miR-199a*-mediated repression of reporter gene activity. Mechanism studies revealed that miR-199a* inhibits Smad1/Smad4-mediated transactivation of target genes, and that overexpression of Smad1 completely corrects miR-199a*-mediated repression of early chondrogenesis. Taken together, miR-199a* is the first BMP2 responsive microRNA found to adversely regulate early chondrocyte differentiation via direct targeting of the Smad1 transcription factor. MicroRNAs (miRNAs) 3The abbreviations used are: miRNA, microRNA; BMP, bone morphogenetic protein; Cbfa1, core binding factor α-1; pRb, retinoblastoma protein; UTR, untranslated region; SBE, Smad-binding elements; Id, inhibitor of DNA binding or inhibitor of differentiation; FZD6, frizzled homolog 6; COMP, cartilage oligomeric matrix protein. are a class of short (∼20–24 nucleotide) non-coding single-stranded RNA molecules that are important regulators of cellular gene expression. First discovered in 1993, they are thought to regulate the expression of approximately one-third of all mammalian genes (1.Lewis B.P. Burge C.B. Bartel D.P. Cell. 2005; 120: 15-20Abstract Full Text Full Text PDF PubMed Scopus (9936) Google Scholar). Functioning at the post-transcriptional level, miRNAs inhibit mRNA expression by binding to the 3′-untranslated region (3′-UTR) of mRNA before directing the repression of translation and/or mRNA degradation. They have been implicated as important regulators of a variety of biological processes including cell proliferation, differentiation, development, and tumorigenesis (2.Chan S.P. Slack F.J. RNA Biol. 2006; 3: 97-100Crossref PubMed Scopus (69) Google Scholar, 3.Chen C.Z. Li L. Lodish H.F. Bartel D.P. Science. 2004; 303: 83-86Crossref PubMed Scopus (2804) Google Scholar, 4.Esau C. Kang X. Peralta E. Hanson E. Marcusson E.G. Ravichandran L.V. Sun Y. Koo S. Perera R.J. Jain R. Dean N.M. Freier S.M. Bennett C.F. Lollo B. Griffey R. J. Biol. Chem. 2004; 279: 52361-52365Abstract Full Text Full Text PDF PubMed Scopus (871) Google Scholar, 5.Harris T.A. Yamakuchi M. Ferlito M. Mendell J.T. Lowenstein C.J. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 1516-1521Crossref PubMed Scopus (856) Google Scholar, 6.Krichevsky A.M. Sonntag K.C. Isacson O. Kosik K.S. Stem Cells. 2006; 24: 857-864Crossref PubMed Scopus (595) Google Scholar, 7.Sempere L.F. Freemantle S. Pitha-Rowe I. Moss E. Dmitrovsky E. 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Biosci. 2009; 14: 2757-2764Google Scholar). miRNAs play an important role in differentiation and development across a whole range of organisms and tissue types. However, little is known about the precise role of miRNAs in cartilage development (10.Lin E.A. Liu C.J. Front. Biosci. 2009; 14: 2757-2764Google Scholar). It is known that miRNAs play a significant role in chondrogenic differentiation, because differential disruption of the Dicer gene in mice results in highly abnormal cartilage development (11.Kobayashi T. Lu J. Cobb B.S. Rodda S.J. McMahon A.P. Schipani E. Merkenschlager M. Kronenberg H.M. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 1949-1954Crossref PubMed Scopus (298) Google Scholar). However, a specific miRNA that regulates chondrogenesis has yet to be identified. There have been various studies on the expression patterns of miRNA in various tissues. For example, miR-140 is exclusively expressed in the cartilage tissue of embryonic zebrafish (12.Berezikov E. Guryev V. van de Belt J. Wienholds E. Plasterk R.H. Cuppen E. Cell. 2005; 120: 21-24Abstract Full Text Full Text PDF PubMed Scopus (1070) Google Scholar). However, direct evidence of the role of miRNA in directing chondrogenic differentiation is lacking. To determine the role of miRNAs in osteochondrogenic differentiation, we developed a miRNA expression profile of mesenchymal stem cells induced by bone morphogenic protein (BMP), by using a microarray approach. Using this profile, we were able to identify potential candidate miRNAs. After screening these candidates, we have obtained experimental evidence to present miR-199a* as a novel miRNA to be directly implicated in the chondrogenic differentiation process, acting as an inhibitor of early chondrogenesis. We were also able to provide evidence that miR-199a* inhibits early chondrogenesis by targeting and suppressing the expression of the Smad protein family 1 (Smad1), a key downstream mediator of BMP signaling and a major regulator of bone and cartilage development (13.Chen D. Zhao M. Harris S.E. Mi Z. Front. Biosci. 2004; 9: 349-358Crossref PubMed Scopus (102) Google Scholar, 14.Chen D. Zhao M. Mundy G.R. Growth Factors. 2004; 22: 233-241Crossref PubMed Scopus (1735) Google Scholar). Construction of Plasmids—The miR-199a* expression plasmid, pSuper199a* was generated using standard DNA techniques. The mouse miR-199a* precursor, including ∼160 bp of genomic flanking sequence, was cloned between the HindIII and XbaI restriction sites of the pSuper-Basic vector (Oligoengine) using the following primer pair: 5′-TTGATAAGCTTCTCCCTGGCCTGTACCATG-3′;5′-GATCTCGAGAAAAATGGTCTGGAAGTTCCCACTG-3′. The primers contained either a HindIII or XhoI (underlined) restriction site. The anti-miR199a*-1 plasmid was generated by cloning a sequence complementary to the hairpin sequence of the precursor miR-199a* molecule. The following primer pair was used: 5′-GATCCCCTCAGGAGGCTGGGACATGTTTCAAGAGAACATGTCCCAGCCTCCTGATTTTTA-3′,5′-AGCTTAAAAATCAGGAGGCTGGGACATGTTCTCTTGAAACATGTCCCAGCCTCCTGAGGG-3′. Because there exists two distinct hairpin precursors for mir-199a*, a second plasmid (anti-miR199a*-2) was generated using the following primer pair: 5′-GATCCCCTCAGGACAATGCCGTTGTATTCAAGAGATACAACGGCATTGTCCTGATTTTTA-3′,5′-AGCTTAAAAATCAGGACAATGCCGTTGTATCTCTTGAATACAACGGCATTGTCCTGAGGG-3′. The control plasmid, pSuper-control was generated by cloning an unrelated miRNA into the pSuper plasmid using the following primer pair: 5′-GATCCCCATGGGTGTGAACCACGAGATTCAAGAGATCTCGTGGTTCACACCCATTTTTTA-3′,5′-AGCTTAAAAAATGGGTGTGAACCACGAGATCTCTTGAATCTCGTGGTTCACACCCATGGG-3′. pGLSmad1, which contains the luciferase reporter gene adjacent to the Smad1 3′-UTR, was generated by cloning the Smad1 3′-UTR sequence into the XbaI site of the pGL3-Control vector (Promega) using the following primer pair: 5′-TAGACTCTAGAAAGACCTGTGGCTTCCGTCTC-3′,5′-CCGACTCTAGAAGTAGAGAAAAACCCTGCTAG-3′. Both primers contained a XbaI restriction site (underlined). Site-directed Mutagenesis—pGLSmad1mut1 and pGLSmad1-mut2 were each derived from pGLSmad1 by mutating one of the two miR-199a* seed sites within the Smad1 3′-UTR sequence (see Fig. 5A for detail). The mutations were carried out using the QuikChange XL Site-directed Mutagenesis kit (Stratagene). Each mutation consisted of replacing four consecutive base pairs at the 3′ region of the seed site. The following primer pair was used for pGLSmad1mut1: 5′-ACGATAATACTTGACCTCTGTGACCATAATTTGGATTGAGAAACTGACAAGCCTTG-3′,5′-CAAGGCTTGTCAGTTTCTCAATCCAAATTATGGTCACAGAGGTCAAGTATTATCGT-3′; and for pGLSmad1mut2: 5′-TTCTGAAACTGTATGCTGGCTGTATATAAGTCAGAATGATGGCAGGCATATGC-3′,5′-GCATATGCCTGCCATCATTCTGACTTATATACAGCCAGCATACAGTTTCAGAA-3′. pGLSmad1mut1,2 was derived from pGLSmad1 by mutating both miR-199a* seed sites, using the primers described above. Total RNA and MicroRNA Isolation for Microarray—Total cellular RNA was purified for microarray analysis by a modified TRIzol reagent (Invitrogen) involving an enhanced precipitation by adding 1 volume of isopropyl alcohol to the extracted aqueous phase, precipitating at -20 °C overnight, and centrifuging the RNA for 30 min at 14,000 × g at 4 °C. For microRNA array profiling, the microRNA population was prepared by the TRIzol method as described above, followed by Flash-PAGE (Ambion) fractionation of total RNA according to the manufacturer's recommendations. The concentration, purity, and integrity of total RNA and fractionated small RNA population were determined by NanoDrop ND-1000 and Agilent 2100 Bioanalyzer. Microarray MicroRNA and mRNA Profiling and Data Analysis—Ambion mirVana TM labeling and the oligonucleotide array system (15.Shingara J. Keiger K. Shelton J. Laosinchai-Wolf W. Powers P. Conrad R. Brown D. Labourier E. RNA (N. Y.). 2005; 11: 1461-1470Crossref PubMed Scopus (239) Google Scholar) were used to monitor expression profiles of ∼380 microRNAs in C2C12 cells, performed in duplicates. GeneSpring GX (Agilent Technologies, Palo Alto, CA) and the TM4 Microarray Software Suite (16.Saeed A.I. Sharov V. White J. Li J. Liang W. Bhagabati N. Braisted J. Klapa M. Currier T. Thiagarajan M. Sturn A. Snuffin M. Rezantsev A. Popov D. Ryltsov A. Kostukovich E. Borisovsky I. Liu Z. Vinsavich A. Trush V. Quackenbush J. BioTechniques. 2003; 34: 374-378Crossref PubMed Scopus (4021) Google Scholar) were used to identify differentially expressed microRNAs in dye-swap experiments. The miRBase Targets (17.Griffiths-Jones S. Saini H.K. van Dongen S. Enright A.J. Nucleic Acids Res. 2008; 36: D154-D158Crossref PubMed Scopus (3648) Google Scholar), TargetScan (18.Lewis 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 (4250) Google Scholar), PicTar (19.Krek 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 (3922) Google Scholar), and miRNAviewer (20.John B. Enright A.J. Aravin A. Tuschl T. Sander C. Marks D.S. PLoS Biol. 2004; 2: e363Crossref PubMed Scopus (2973) Google Scholar) miRNA target prediction algorithms were used to identify microRNA targets. Real-Time PCR—Total RNA (including total microRNA) was harvested from C3H10T1/2 cells using the mirVana miRNA isolation kit (Ambion). The RNA was reverse transcribed using the TaqMan miRNA reverse transcription kit (Applied Biosystems) and miRNA-specific primers (Applied Biosystems). MicroRNA expression levels were then analyzed using the appropriate TaqMan miRNA assay (Applied Biosystems) as per the manufacturer's instructions. Quantitation of the ubiquitously expressed miRNA, snoRNA202, was performed as an endogenous control. To determine the expression levels of Col2a1 and cartilage oligomeric matrix protein (COMP), total RNA was analyzed using the ImProm-II Reverse Transcription System (Promega) followed by real-time PCR with SYBR Green chemistry. A reaction mixture containing the SYBR Green Master Mix (Applied Biosystems) and the appropriate primers was added to a 96-well plate, together with 1 μl of cDNA template, for a final reaction volume of 20 μl/well, and run for an initial step at 95 °C for 10 min, followed by 40 cycles of amplification at 95 °C for 15 s and 60 °C for 60 s. Absolute quantization of glyceraldehyde-3-phosphate dehydrogenase was performed as an endogenous control. All real-time PCR were carried out in an ABI prism 7500 PCR amplification system (PE Biosystems, Foster City, CA). Primer sequences are as follows: glyceraldehyde-3-phosphate dehydrogenase forward, 5′-ATGACATCAAGAAGGTGGTG-3′, glyceraldehyde-3-phosphate dehydrogenase reverse, 5′-CATACCAGGAAATGAGCTTG-3′; Col2a1 forward, 5′-TGGTGGAGCAGCAAGAGCAA-3′, Col2a1 reverse, 5′-CAGTGGACAGTAGACGGAGGAAA-3′; Smad1 forward, 5′-GCTTCGTGAAGGGTTGGGG-3′, Smad1 reverse, 5′-CGGATGAAATAGGATTGTGGGG-3′. Reporter Gene Assay—Inhibition of Smad1 reporter constructs was assayed by co-transfection of C3H10T1/2 cells with pGL3Smad1 (1.5 μg), various amounts of pSupermir-199a*, and a pSVGal control plasmid (1.5 μg). Additionally, pSuper vector was used to bring the total amount of transfected DNA to 11 μg. Co-transfections were carried out using Lipofectamine 2000 (Invitrogen) in 6-well plates as per the manufacturer's instructions. Cultures were harvested after 48 h of incubation. Luciferase and β-galactosidase activities were assayed using the Tropix Galacto-Star Reporter Gene Assay system (Applied Biosystems) and Luciferase Assay Regent (Promega). Measurements were carried out in a Mini-Lum Luminometer (Bioscan). The assays were performed in triplicate. Immunoblotting Analysis—To examine the expression of Sox9, Col2A1, and COMP in the course of chondrogenesis, total cell extracts prepared from micromass cultures of C3H10T1/2 cells were mixed with 5 × sample buffer (312.5 mm Tris-HCl (pH 6.8), 5% β-mercaptoethanol, 10% SDS, 0.5% bromphenol blue, 50% glycerol). Proteins were resolved on a 10% SDS-polyacrylamide gel and electroblotted onto a nitrocellulose membrane. After blocking in 10% nonfat dry milk in Tris-buffered saline, Tween 20 (10 mm Tris-HCl (pH 8.0), 150 mm NaCl, 0.5% Tween 20), blots were incubated with anti-Sox9, anti-COMP, or anti-Col 2 antibody (diluted 1:1000) for 1 h. Anti-tubulin antibody (diluted 1:2000) was used as a loading control. After washing, the respective secondary antibody (horseradish peroxidase-conjugated anti-rabbit immunoglobulin; 1:1000 dilution) was added, and bound antibody was visualized using an enhanced chemiluminescence system (Amersham Biosciences). Chondrogenic Differentiation Assay—Cultures of C3H10T1/2 murine mesenchymal stem cells were maintained in standard 10-cm tissue culture dishes in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, with a change of medium every 2–3 days. Cultures were stored in a humidified incubator at 37 °C and 5% CO2. A modified micromass culture technique was carried out, as previously described (21.Liu C. J. Musculoskelet. Neuronal Interact. 2005; 5: 340-341PubMed Google Scholar, 22.Liu C.J. Prazak L. Fajardo M. Yu S. Tyagi N. Di Cesare P.E. J. Biol. Chem. 2004; 279: 47081-47091Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 23.Liu T. Gao Y. Sakamoto K. Minamizato T. Furukawa K. Tsukazaki T. Shibata Y. Bessho K. Komori T. Yamaguchi A. J. Cell. Physiol. 2007; 211: 728-735Crossref PubMed Scopus (101) Google Scholar). Transfections were carried out using Lipofectamine 2000 (Invitrogen) in 6-well plates as per the manufacturer's instructions. For each sample, 4 μg of plasmid and 10 μl of Lipofectamine 2000 was diluted with serum-free Dulbecco's modified Eagle's medium to reach a total volume of 500 μl. After incubation for 20 min at room temperature, the mixture was introduced into the well containing cells growing at ∼90% confluence in 2 ml of Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Following transfection with the specified plasmids, the cells were incubated for 48 h. The cells were then trypsinized and brought into suspension at a density of 107 cells/ml. 10 μl of the suspension was placed into the center of each well on a standard 12-well polystyrene tissue culture plate. After incubation for 2 h at 37 °C and 5% CO2, wells were flooded with 1 ml of culture medium (supplemented with fetal bovine serum) containing 100 ng/ml recombinant BMP-2. Replacement with fresh medium containing BMP-2 was carried out every 2–3 days. Mouse prechondrogenic ATDC5 cells were maintained in a medium consisting of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium (Flow Laboratories, Irvine, UK) containing 5% fetal bovine serum (Invitrogen), 10 μg/ml human transferrin (Roche Applied Science), and 30 nm sodium selenite (Sigma) at 37 °C in a humidified atmosphere of 5% CO2 in air. ATDC5 cells transfected with either pSuper control encoding a nonrelated miRNA, pSuper-mir199a* or pSuper-anti-mir199a* plasmid, were seeded at a density of 3 × 105 cells per well in 6-well cell-culture plates (Corning, Slangerup, Denmark). To induce chondrogenesis, cells were cultured in the medium supplemented with 10 μg/ml human insulin (Sigma) (24.Chen L. Fink T. Zhang X.Y. Ebbesen P. Zachar V. Differentiation. 2005; 73: 350-363Crossref PubMed Scopus (36) Google Scholar, 25.Kong L. Liu C.J. Cell. Mol. Life Sci. 2008; 65: 3494-3506Crossref PubMed Scopus (15) Google Scholar, 26.Shukunami C. Shigeno C. Atsumi T. Ishizeki K. Suzuki F. Hiraki Y. J. Cell Biol. 1996; 133: 457-468Crossref PubMed Scopus (346) Google Scholar). The medium was replaced every 2–3 days. Within 3 days, both mRNA and protein of cells were collected for real-time PCR and Western blot assay. miRNAs Are Differentially Expressed following BMP-2 Treatment of C2C12 Cells—To identify candidate miRNAs that regulate bone and/or cartilage differentiation and development, the expression profile of numerous miRNAs was determined via commercial microarray. C2C12 cells were exposed to BMP-2 (200 ng/ml) and harvested within 24 h; total RNA was extracted and used for array analysis. miRNA expression patterns following BMP-2 treatment were then compared with reference levels obtained from cells prior to BMP-2 induction. Microarray analysis revealed multiple miRNAs that were either positively or negatively regulated by BMP-2 induction (Fig. 1A). Based on these results, the expression levels of selected miRNAs were assayed using real-time PCR in C2C12 cells at various time points following BMP-2 induction. The expression levels of miR-374, miR-210, and miR-143 were largely consistent with the microarray data (Fig. 1, B–D). Interestingly, although the microarray showed the expression of miR-374 and miR-143 to be increased and decreased, respectively, the TaqMan real-time PCR assay revealed that both miRNAs had similar expression profiles, characterized by a significant increase in expression during the first 12 h of BMP-2 induction, followed by a rapid decline to extremely low expression levels (Fig. 1, B and D). The microarray data showed that miR-199a* was significantly up-regulated at 24 h following BMP-2 induction, with expression levels that were ∼4-fold greater than those in untreated cells. miR-199a* is highly expressed in the embryonic skeletal system of a wide range of organisms (27.Ason B. Darnell D.K. Wittbrodt B. Berezikov E. Kloosterman W.P. Wittbrodt J. Antin P.B. Plasterk R.H. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 14385-14389Crossref PubMed Scopus (212) Google Scholar, 28.Landgraf P. Rusu M. Sheridan R. Sewer A. Iovino N. Aravin A. Pfeffer S. Rice A. Kamphorst A.O. Landthaler M. Lin C. Socci N.D. Hermida L. Fulci V. Chiaretti S. Foa R. Schliwka J. Fuchs U. Novosel A. Muller R.U. Schermer B. Bissels U. Inman J. Phan Q. Chien M. Weir D.B. Choksi R. De Vita G. Frezzetti D. Trompeter H.I. Hornung V. Teng G. Hartmann G. Palkovits M. Di Lauro R. Wernet P. Macino G. Rogler C.E. Nagle J.W. Ju J. Papavasiliou F.N. Benzing T. Lichter P. Tam W. Brownstein M.J. Bosio A. Borkhardt A. Russo J.J. Sander C. Zavolan M. Tuschl T. Cell. 2007; 129: 1401-1414Abstract Full Text Full Text PDF PubMed Scopus (3045) Google Scholar, 29.Wienholds E. Kloosterman W.P. Miska E. Alvarez-Saavedra E. Berezikov E. de Bruijn E. Horvitz H.R. Kauppinen S. Plasterk R.H. Science. 2005; 309: 310-311Crossref PubMed Scopus (1345) Google Scholar). Therefore, using real-time PCR TaqMan® miRNA assays, we sought to verify the expression pattern of miR-199a* following BMP-2 treatment in C3H10T1/2 cells, a well established in vitro cell model for studying chondrogenesis (22.Liu C.J. Prazak L. Fajardo M. Yu S. Tyagi N. Di Cesare P.E. J. Biol. Chem. 2004; 279: 47081-47091Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 30.Liu C.J. Zhang Y. Xu K. Parsons D. Alfonso D. Di Cesare P.E. Front. Biosci. 2007; 12: 3899-3910Crossref PubMed Scopus (44) Google Scholar). At 6 h following BMP-2 treatment, the expression of miR-199a* was reduced below the limit of detection of the assay (Fig. 1E); however, at 24 h, miR-199a* levels had risen dramatically and continued to rise gradually over the following 6 days. The highest level of expression occurred on the 7th day of BMP-2 induction and was ∼2-fold greater than that for untreated C3H10T1/2 cells. In addition, to verify our results in other miRNAs, we also analyzed the expression pattern of miR-374 in BMP-2-treated C3H10T1/2 cells. Such analysis showed miR-374 levels to be significantly increased following BMP-2 treatment, as expected (Fig. 1F). miR-199a* Overexpression Inhibits Chondrogenic Differentiation—To determine whether miR-199a* mediates chondrogenic differentiation, we constructed the miRNA expression plasmid, pSuper199a*, by cloning the miR-199a* precursor into the pSuper vector, followed by transfection into C3H10T1/2 cells, using techniques described previously (31.Shi B. Sepp-Lorenzino L. Prisco M. Linsley P. deAngelis T. Baserga R. J. Biol. Chem. 2007; 282: 32582-32590Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Real-time PCR analysis of C3H10T1/2 cells transfected with pSuper199a* revealed a significant decrease in the mRNA expression levels of chondrogenesis markers Col2A1 and COMP, when compared with control cells 24 h after induction with BMP-2 (100 ng/ml) (Fig. 2A). This suggests that miR-199a* functions as an inhibitor of the early stages of chondrogenic differentiation. Because the cell models used thus far have relied on BMP-2 to induce chondrogenesis, we next sought to determine whether the effects of miR-199a* were specific to the BMP-2 chondrogenic growth factor. To do so, we repeated the experiment using murine prechondrogenic ATDC5 cells that undergo chondrogenic differentiation in the presence of human insulin (10 mg/ml). We compared cells transfected with pSuper199a* or a control plasmid encoding an unrelated miRNA, pSuper-control. Both immunoblotting analysis (Fig. 2B) and real-time PCR (Fig. 2C) revealed a significant decrease in early chondrogenesis markers Col2A1, COMP, and Sox9 in this ATDC5 cell model. This suggests that miR-199a* functions as a mediator of early chondrogenesis, and not as a general response to BMP-2 signaling. Inhibition of miR-199a* Enhances Chondrogenic Differentiation—To further define the role of miR-199a* in chondrogenic differentiation, we constructed a miRNA expression plasmid, anti-miR199a*-1, that generated a short RNA sequence complementary to the hairpin sequence of the miR-199a* precursor molecule. By binding to the miR-199a* precursor, the products of this plasmid would inhibit miR-199a* synthesis. Because two distinct precursors have been identified for miR-199a*, a second plasmid, anti-miR-199a*-2 was also generated. ATDC5 cells were transfected with either an equimolar mixture of anti-miR199a*-1 and anti-miR199a*-2 or pSuper-Control and chondrogenesis was induced. Both real-time PCR and Western blotting assays revealed a significant increase in chondrogenesis markers Col2A1, COMP, and Sox9, when compared with the control (Fig. 3, A and B). Smad Family 1 (Smad1) Is a Target of miR-199a*—The miRNA target prediction program miRanda was utilized to generate a list of predicted mRNA targets for miR-199a*. This list was then used to identify putative miRNA target genes that are coincidentally involved in BMP-2 signaling and chondrogenic differentiation. Members of the miR-199 family were predicted to target members of the Smad gene family. Smad1 and Smad5, which are known downstream mediators of BMP signaling in osteochondroprogenitor cells (3.Chen C.Z. Li L. Lodish H.F. Bartel D.P. Science. 2004; 303: 83-86Crossref PubMed Scopus (2804) Google Scholar), were both identified as putative targets of miR-199a* (Fig. 4A). Smad1 is an established mediator of both osteoblastic and chondrogenic differentiation of progenitor cells in response to stimulation by BMPs (13.Chen D. Zhao M. Harris S.E. Mi Z. Front. Biosci. 2004; 9: 349-358Crossref PubMed Scopus (102) Google Scholar, 14.Chen D. Zhao M. Mundy G.R. Growth Factors. 2004; 22: 233-241Crossref PubMed Scopus (1735) Google Scholar, 32.Hatakeyama Y. Nguyen J. Wang X. Nuckolls G.H. Shum L. J. Bone Joint Surg. Am. 2003; 85: 13-18Crossref PubMed Scopus (56) Google Scholar). The putative repression of Smad1 mRNA expression by miR-199a* would hinder BMP-2 signaling, and thus serve to explain how overexpression of miR-199a* may inhibit BMP-2-induced chondrogenic differentiation. miRNA inhibits mRNA expression by binding to a specific sequence with the 3′-UTR, also known as a “seed site.” Perfect complementarity of a particular miRNA to the seed site is thought to result in mRNA cleavage, whereas imperfect complementarity generally results in translational repression, without degrading the mRNA itself (33.Carrington J.C. Ambros V. Science. 2003; 301: 336-338Crossref PubMed Scopus (1511) Google Scholar, 34.Doench J.G. Petersen C.P. Sharp P.A. Genes Dev. 2003; 17: 438-442Crossref PubMed Scopus (1016) Google Scholar, 35.Zeng Y. Yi R. Cullen B.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9779-9784Crossref PubMed Scopus (726) Google Scholar). miR-199a* binds to a seed site within the Smad1 3′-UTR with near, but not perfect complementarity (Fig. 5A), suggesting that it may not affect the level of Smad1 mRNA. However, imperfect complementarity can also result in mRNA cleavage, in certain cases (36.Kim V.N. Genes Dev. 2006; 20: 1993-1997Crossref PubMed Scopus (206) Google Scholar, 37.Kim V.N. Nam J.W. Trends Genet. 2006; 22: 165-173Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar, 38.Yekta S. Shih I.H. Bartel D.P. Science. 2004; 304: 594-596Crossref PubMed Scopus (1414) Google Scholar). In addition, the Smad1 3′-UTR contains two putative miR-199a* seed sites, which would suggest a stronger association than if only one site were present (Fig. 4B). To determine whether miR-199a* cleaves Smad1 mRNA, the expression of Smad1 mRNA was measured via real-time PCR immediately following BMP-2 induction of C3H10T1/2 cells. The pattern of Smad1 mRNA expression was found to be th
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