miR-29 in Myoblast Conversion to Myofibroblast Figure 6. A model of TGF-b-Smad3-miR-29 circuit in myogenic and fibrogenic differentiation of C2C12 myoblasts. The model depicts the roles of the TGF-b-Smad3-miR-29 regulatory circuit in myogenic and fibrogenic differentiation of C2C12 cells. In the normal myogenesis, the recruitment of MyoD/SRF and the displacement of YY1/PRC from miR-29 promoter lead to the elevation of miR-29 expression and its feedback inhibition on YY1 and successful myogenic differentiation. Upon TGF-b stimulation, activated Smad3 translocates into nucleus where it binds to SBE, resulting in MyoD dissociation as well as YY1/Ezh2 recruitment to multiple CCAT boxes of miR-29b/c promoter. This leads to trimethylation of histone lysine 27 and subsequent silencing of miR-29 expression. Loss of miR-29 upregulates the expression of ECM genes such as Collagens as well as cell adhesion genes such as Lims1, thus promoting the conversion of myoblasts into myofibroblasts. Straight line, promoter/enhancer region of mmu- miR-29b/c with arrow denotes TSS; CCAT, YY1 binding elements; E-box, MyoD binding sites; SBE, Smad3 binding element; Me, methylation of histone lysine 27; Ac, acetylation of histones. doi:10.1371/journal.pone.0033766.g006 glutamine, 100 U/ml penicillin, and 100 mg of Streptomycin at 37 C in 5% CO2. For myofibroblast transdifferentiation and myogenic differentiation experiments, cells were seeded in 60 mm or 100 mm plates and when 90% confluent they were shifted to DMEM without FBS containing 2% horse serum. Cells were treated with 5 ng/ml TGF-b1 (R&D systems). 10T1/2 cells and HEK 293T cells were cultured in DMEM supplemented with 10% FBS. Transfections and infections Transient transfections with miRNA precursor oligos and siRNA oligos or DNA plasmids were performed in 60 mm or 100 mm dishes with Lipofectamine 2000 reagent as suggested by the manufacturer (Invitrogen). For luciferase experiments, C2C12 and primary myoblasts were transfected in 12-well plates. Cell extracts were prepared and luciferase activity was monitored as previously described [36] or using dual-luciferase kit (Promega). To produce virus particles expressing vector or miR-29, pMIF- cGFP-Zeo Vector or pMIF-cGFP-Zeo-miR-29 plasmids along with the packaging plasmid mix (pPACK) (System Biosciences) were transfected into HEK293T cells maintained in 10% FBS. 48 h after transfection, supernatant was harvested from these cells and viral titers were estimated by FACS analysis. Approximately 16109 virus particles were used to transduce C2C12 cells, which were subsequently placed in 400 mg/ml Zeocin for stable selection. Stable clones were pooled together after ,2 week selection. Oligonucleotides Precursor miRNA oligos were obtained from Ambion. Mercury LNA microRNA or control oligos were obtained from Exiqon. The 19-nucleotide siRNA duplexes against mouse Smad 2 coding region (siRNA, 59-GAAUUGAGCCACAGAGUAA-39), Smad 3 coding region (siRNA, 59-CAGUUCUACCUCCAGUGUU-39) or Smad 7 coding region (siRNA, 59-GCACUCGGUGCUCAA- GAAA-39) or scrambled oligos were obtained from Ribobio. In each case 50 mM oligos were used for transient transfections into cells. DNA constructs Col 1A1-3UTR, Col 1A2-3 UTR, Col 3A1-3 UTR luciferase reporter plasmids were kind gifts from Dr. Ahlquist. Paul [37]. For the construction of mutant plasmid, the 29 base pair seed region of the predicted miR-29 binding site was deleted from the above parental constructs using QuickChange XL-mutagenesis (Strata- gene). To construct Lims1-39UTR reporter plasmid, a 45 bp fragment encompassing miR-29 binding site was cloned into pMIR-report vector (ABI) between Spa1 and Sac1 sites. Mutant reporter plasmids were generated by mutating the seed region from TGGTGCT to TACCTCT. Replication-deficient lentivirus- based expression plasmids pMIF-cGFP-Zeo Vector and pMIF- cGFP-Zeo-miR-29b, along with the packaging plasmid mix (pPACK), were obtained from System Biosciences (SBI). An YY1 expression plasmid was a gift from Y. Shi (Harvard PLoS ONE | www.plosone.org 11 March 2012 | Volume 7 | Issue 3 | e33766