miR-29 in Myoblast Conversion to Myofibroblast Figure 4. Smad3 mediates the repression of TGF-b on miR-29 at the transcriptional level by binding to miR-29 promoter region. (A) C2C12 myoblasts were transfected with siRNA oligos against Smad2, Smad3 or Smad7, using scrambled siRNAs (SCR) as a control. 48 hr post- transfection, the expressions were examined by Western blotting using a-Tubulin as a loading control. (B) C2C12 myoblasts, transfected with the indicated siRNA oligos, were incubated with TGF-b in DM for 48 hrs at which time the expressions of miR-29 were measured. (C) miR-29-promoter-luc reporter activities in C2C12 myoblasts transfected with the above siRNA oligos and then treated with TGF-b in DM for 48 hrs. (D) miR-29 expressions in primary myoblasts isolated from Smad3+/+, Smad3+/2 or Smad32/2 mice. (E) miR-29 expressions in primary myoblasts from Smad7+/+ or Smad72/2 mice. (F) Smad7+/+ or Smad72/2 mice were injected with Cardiotoxin (CTX) into TA muscles to induce muscle regeneration. Muscles were harvested at designated days after the injection and assayed for miR-29 expression levels by qRT-PCR. (G) C2C12 myoblasts were untreated or treated with TGF-b for 12 hrs at which time chromatins were collected for ChIP assays using antibodies against Smad3 or IgG as controls. PCR assays were then used to measure the enrichment fold of Smad3 on three putative binding sites, S1, S2 and S3. doi:10.1371/journal.pone.0033766.g004 demonstrate that RNA-seq represents a powerful new tool to determine the overall cascade of events under influence by miRNA. Its broader dynamic range allows the analysis of both high- and low-abundance transcripts and facilitates the analysis of genes spanning a wide spectrum of expression levels. Our results reveal that in addition to promoting myogenic differentiation, miR-29 inhibits the expression of a large number of ECM genes including Collagens, MATN1, ECM1 (Tables S2 and S4). This is in line with others’ results and led us to believe that miR-29 inhibits the transdifferentiation of myoblasts into myofibroblasts. In addition to ECM genes, we found that cell adhesion genes represent an important category of genes under control by miR- PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e33766