miR-29 in Myoblast Conversion to Myofibroblast Figure 1. Stable over-expression of miR-29 in C2C12 cells down-regulates ECM and cell adhesion genes. (A) Differentially expressed genes between C2C12 cells stably expressing vector negative control (NC) and miR-29 were determined by RNA-seq. X- and Y-axis represent the log2 based FPKM values for expressed genes in NC and miR-29 samples, respectively. The black dots represent genes with no significant expression changes between NC and miR-29 samples. The red dots represent genes with significant expression changes. The blue dots represent genes that have expression signal in only one sample but absent in the other. (B) Over-represented GO terms by GO analysis of down-regulated list of genes. CC: cellular component; BP: biological process. SP_PIR: a database of protein super-family names; KEGG: Kyoto Encyclopedia of Genes and Genomes. (C) Coverage plot showing a ,20 kb region encompassing the Collagen 1A1 (Col) gene on chromosome (Chr) 11; the gene structure is shown in blue below the graph. doi:10.1371/journal.pone.0033766.g001 a-SMA and VIM expression indirectly. Moreover, knock-down of miR-29 led to opposite augmenting effect on Col 1A1, Col 1A2 and Col 3A1 expression (Fig. 2D), supporting that collagens are direct targets of miR-29. This notion was further examined by using reporters with a fragment of the collagen (Col 1A1, Col 1A2, Col 3A) 39 UTR containing the miR-29 binding site fused downstream of the firefly luciferase (Luc) gene. Co-transfections of the reporter plasmid (WT) with miR-29 caused significant repressions of luciferase activities (Fig. 2E). This regulation appeared specific to miR-29 binding since changes in luciferase activity were not impacted when transfections were repeated with an irrelevant miRNA, miR-212, or with the miR-29 site deleted from the collagen 39UTR (Mutant). In addition to miR-29c, the other two members of miR-29 family, miR-29a and miR-29b could also target Collagen 39UTR (Fig. S2). Collectively, our findings suggest that high level of miR-29 is important for driving myogenic differentiation and loss of miR-29 promotes transdiffer- entiation of myoblasts into myofibroblasts by targeting Collagens. PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e33766