In addition to its pro-fibrogenic roles, TGF-b is well- characterized as a potent inhibitor of myogenic differentiation. Smad3 has been shown to physically interact with MRFs to repress their transcriptional activity. In particular, Smad3, but not Smad2, blocks MyoD-mediated transcriptional activation by associating with bHLH region of MyoD. This interaction interferes with MyoD/E protein dimerization and cooperative binding to E-boxes [12]. Very recently, interplay between TGF-b and miR-29 was discovered in the regulation of myogenic differentiation [13]. TGF-b treatment suppressed the expression of miR-29 which in turn up-regulates Histone Deacetylase 4 (HDAC4) to inhibit the myogenic commitment. However, it was not clear how TGF-b exerts the suppression on miR-29. We therefore sought to determine whether it is at the transcriptional level through Smad3 and what other factors are involved. Although it is not known whether miR-29 plays a part in regulating transdifferentiation of myoblasts into myofibroblasts, emerging studies implicated miR-29 family in cardiac, liver, pulmonary, skin and muscle fibrosis [14,15,16,17,18,19]. Multiple ECM genes such as collagens, fibrillins and elastin [15,16,17,18,19] are identified as direct targets of miR-29 in fibroblasts, implicating miR-29 as a potent factor in modulating ECM modeling and tissue fibrosis. It was shown that intramus- cular injection of miR-29 into dystrophic muscles down-regulated collagen expression [19]; however, the cellular mechanisms underlying this anti-fibrotic action of miR-29 was still obscure. Furthermore, it was not clear whether miR-29 regulates both the anti-myogenic and the pro-fibrogenic effect of TGF-b signaling. We thus investigated the possible involvement of miR-29 during the conversion of myoblasts into myofibroblasts as well as its interaction with TGF-b/Smad3 signaling. In this study, in an effort trying to gain insights into the global effect of miR-29 on myogenic cells, a transcriptome analysis by high throughput RNA-sequencing (RNA-seq) was conducted and the results revealed that miR-29 down-regulates ECM and cell adhesion genes in addition to promoting the myogenic differen- tiation, suggesting a role of miR-29 in suppressing fibrogenic differentiation of myoblasts. Subsequent analyses demonstrated that indeed miR-29 inhibits C2C12 transdifferentiation into myofibroblasts by suppressing both collagens and Lims1 (LIM and senescent cell antigen-like-containing domain protein). Furthermore, we demonstrated that TGF-b controls both the pro-myogenic and anti-fibrogenic functions of miR-29. The inhibition of miR-29 by TGF-b was mediated by Smad3 at the transcriptional level through both inhibiting MyoD binding and enhancing YY1/Polycomb recruitment on its promoter region. Results Analysis of miR-29 affected transcriptome using RNA-seq In order to gain insights into the miR-29 mediated events in muscle cells, we decided to conduct a genome-wide transcriptome analysis. A C2C12 cell line stably expressing miR-29 was established by infecting cells with a miR-29 expressing lentivirus. Analogous to transiently transfected cells [5], these cells differen- tiated faster than vector transfected negative control (NC) cells (data not shown). Subsequently, PolyA-tailed mRNAs from control and miR-29 cells were subjected to transcriptome analysis using a RNA-seq platform. Compared to traditional microarray-based analysis of transcriptomes, mRNA-seq provides higher level of accuracy and broader dynamic ranges and has been proven to be suitable for assessing the relatively moderate influences that miRNAs have on their target mRNAs [20]. A total of 26.3 million and 11.4 million raw reads were sequenced from miR-29 miR-29 in Myoblast Conversion to Myofibroblast and NC samples, respectively, which were then mapped to mouse NCBIM37.61 mm9 reference genome via Tophat v1.2 [21]. The majority of reads can be mapped to exonic regions (.10 FPKM) and much fewer (,0.05 FPKM) in introns and non-coding regions (Fig. S1), indicating great specificity for expressed mRNA and rejection of genomic DNA and unspliced pre-mRNA. Cuffdiff program from Cufflinks package (v1.0.0) [22] was subsequently employed to identify the differentially expressed genes under a false discovery rate (FDR) of 5%. As a result, a total of 472 and 739 genes were found to be up- and down-regulated in miR-29 expressing cell line vs NC cell line (Fig. 1A and Table S1 and Table S2). Subsequent Gene Ontology (GO) analysis with up- regulated list of genes revealed that the top ranked lists of enriched GO categories include ‘‘contractile fiber’’, ‘‘contractile fiber part’’, ‘‘sarcomere’’, ‘‘myofibril’’, ‘‘I band’’, ‘‘Z disc’’ (Table S3), which is in agreement with the previously identified roles of miR-29 in accelerating muscle regeneration. Strikingly, GO analysis with down-regulated list of genes revealed an over-representation of ECM genes presented in GO categories such as ‘‘Extracellular matrix’’, ‘‘Extracellular matrix part’’, ‘‘Proteinaceous extracellular matrix’’, ‘‘Collagen’’ et al. (Fig. 1B–C, Fig. S1C and Table S4). This is in agreement with the emerging reports demonstrating the pivotal role of miR-29 in ECM remodeling as well as fibrosis of multiple tissues [15,16,17,18] (Fig. S1D–E). In addition, we noticed that cell-adhesion genes under GO categories ‘‘Cell adhesion’’, ‘‘Biological adhesion’’ and ‘‘Focal adhesion’’ represent another category of genes under significant influence by miR-29 expression (Fig. 1B). miR-29 suppresses C2C12 myoblast conversion into myofibroblast through targeting Collagens and Lims1 The decrease of ECM expression in miR-29 expressing cells led us to hypothesize that miR-29 functions to inhibit the transdiffer- entiation of C2C12 myoblasts into fibrogenic cells. We reasoned that myoblasts have the potential to transdifferentiate into myofibroblasts. However, under normal myogenic differentiation condition, YY1 regulated miR-29 drives myoblasts fusion into myotubes [5], suppressing the fibrogenic pathway. To test this notion, the expression levels of fibrotic markers during C2C12 differentiation were evaluated. qRT-PCR analysis data presented in Figure 2A showed an up-regulation of Col 1A1, 1A2, Col 3A1 and a-SMA (alpha Smooth Actin) during early myoblast differentiation (DM 1d), which is in line with the previous findings from a transcriptional profiling of gene expression changes during C2C12 differentiation [23]. The early rise of these genes probably reflected the need of ECM molecules for cell adhesion, motility, spreading, and anchorage-dependent growth at the early stage of differentiation. However, the expression levels were significantly down-regulated in late times of differentiation (day 2 and 4) concomitant with the up-regulation of myofibrillar genes, Myosin Heavy Chain (MyHC), alpha Skeletal Actin (a-Actin), Troponin and Myogenin, suggesting that fibrogenic trans-differentiation of C2C12 cells was inhibited during terminal myogenic differentia- tion. In order to assess whether miR-29 is a critical factor in determining the fate of myoblast differentiation, miR-29 was over- expressed in C2C12. As anticipated, the myogenic differentiation was accelerated as assessed by increased expression levels of Myogenin, MyHC, Troponin and a-Actin (Fig. 2B). However, the expressions of Col 1A1, Col 1A2, and Col 3A1 were suppressed (Fig. 2C), suggesting that miR-29 inhibits fibrogenic differentiation likely through targeting collagens. Interestingly, a-SMA and VIM were also found to be down-regulated although they are not predicted to be direct targets of miR-29 by multiple computational algorithms (data not shown), indicating that miR-29 may control PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e33766