Inhibition of miR-29 by TGF-beta-Smad3 Signaling through Dual Mechanisms Promotes Transdifferentiation of Mouse Myoblasts into Myofibroblasts Liang Zhou1,3, Lijun Wang1,3, Leina Lu2,3, Peiyong Jiang2,3, Hao Sun2,3*, Huating Wang1,3* 1 Department of Obstetrics and Gynaecology, Chinese University of Hong Kong, Hong Kong, China, 2 Department of Chemical Pathology, Chinese University of Hong Kong, Hong Kong, China, 3 Li Ka Shing Institute of Health Sciences, Chinese University of Hong Kong, Hong Kong, China Abstract MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression in post-transcriptional fashion, and emerging studies support their importance in regulating many biological processes, including myogenic differentiation and muscle development. miR-29 is a promoting factor during myogenesis but its full spectrum of impact on muscle cells has yet to be explored. Here we describe an analysis of miR-29 affected transcriptome in C2C12 muscle cells using a high throughput RNA-sequencing platform. The results reveal that miR-29 not only functions to promote myogenic differentiation but also suppresses the transdifferentiation of myoblasts into myofibroblasts. miR-29 inhibits the fibrogenic differentiation through down-regulating both extracellular matrix genes and cell adhesion genes. We further demonstrate that miR-29 is under negative regulation by TGF-beta (TGF-b)–Smad3 signaling via dual mechanisms of both inhibiting MyoD binding and enhancing Yin Yang 1 (YY1)-recruited Polycomb association. Together, these results identify miR-29 as a pleiotropic molecule in both myogenic and fibrogenic differentiation of muscle cells. Citation: Zhou L, Wang L, Lu L, Jiang P, Sun H, et al. (2012) Inhibition of miR-29 by TGF-beta-Smad3 Signaling through Dual Mechanisms Promotes Transdifferentiation of Mouse Myoblasts into Myofibroblasts. PLoS ONE 7(3): e33766. doi:10.1371/journal.pone.0033766 Editor: Donald Gullberg, University of Bergen, Norway Received November 26, 2011; Accepted February 21, 2012; Published March 16, 2012 Copyright: 2012 Zhou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work described in this paper was substantially supported by three General Research Funds (GRF) from the Research Grants Council (RGC) of the Hong Kong Special Administrative Region, China (CUHK476309 and 476310 to HW and 473211 to HS), two CUHK direct grants to HW (2041492 and 2041662) and a CUHK Direct Grant to HS (2041474). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: haosun@cuhk.edu.hk (HS); huating.wang@cuhk.edu.hk (HW) Introduction microRNAs (miRNAs) are non-coding single-stranded RNAs of 21–25 nucleotides and constitute a novel class of gene regulators that are found in a variety of eukaryotic organisms. miRNAs negatively regulate their targets at the post-transcriptional level through binding to their 39 UTRs [1,2]. Mounting evidences support the importance of miRNAs in skeletal muscle development and muscle related diseases. The process of skeletal muscle cell differentiation is orchestrated by transcription factors MyoD, Myf5, myogenin, MRF4, and Mef2. These factors activate muscle genes to coordinate myoblasts to terminally withdraw from cell cycle and subsequently fuse into multinucleated myotubes [3]. A handful of miRNAs were studied in muscle system and proven to be critical in regulating myogenic differentiation [4]. Previously, our group identified miR-29 as a pro-myogenic factor [4,5]. In undifferentiated myoblasts, miR-29 expression is epigenetically silenced by a repressive complex containing Yin Yang 1 (YY1) and Polycomb protein, Enhancer of Zeste Homolog 2 (Ezh2), which is associated to the miR-29 promoter region causing tri-methylation of histone 3 lysine 27 (H3K27me3). As differentiation ensues, MyoD replaces the silencing complex causing the derepression of miR-29 transcriptional expression. In turn, the accumulation of miR-29 during differentiation leads to the depletion of YY1 which is also a repressor of muscle genes. We further demonstrated that this regulatory circuit is disrupted in Rhabdomyosarcoma which may contribute to the development of this tumor. These findings suggest that miR-29 involved circuitries are critical regulator of gene expression in skeletal muscle cells. Thus, it is our interest to explore the full spectrum of the influence by miR-29 in these cells and discover other targets under the control of miR-29. In addition to the normal myogenic differentiation, muscle myogenic cells possess the potential to transdifferentiate into other mesenchymal lineages. For example, Bone Morphogenic Protein (BMP) signaling triggers C2C12 transdifferentiation into osteoblasts whereas PPARgamma (PPARc) promotes its adipogenic transdif- ferentiation [6,7]. Of particular interest, transdifferentiation of myogenic cells into myofibroblasts was thought to contribute to the accumulation of Extracellular Matrix (ECM) molecules and the onset of fibrosis in injured skeletal muscle [8,9]. TGF-beta (TGF-b), one of the most potent fibrogenic cytokines, has been individuated as the major inducer of transdifferentiation of myogenic cells into myofibroblasts as well as muscle fibrogenesis [8,9,10,11]. After binding to the receptors, TGF-b phosphorylates and activates downstream mediators, mainly Smad2 and Smad3, inducing their translocation to the nucleus, where they regulate the expression of many target genes, including fibrotic genes, through binding to the Smad Binding Element (SBE) on their promoter/enhancer. In addition, TGF-b can induce its downstream inhibitory Smad7, which in turn inhibits Smad2/3 phosphorylation via the negative feedback mechanisms. The underlying mechanisms mediating the pro-fibrogenic effect of TGF-b in C2C12 cells were not fully understood. Both Rho kinase signaling and Notch2 have been shown to be downstream mediators [10,11]. PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e33766