miR-29 in Myoblast Conversion to Myofibroblast Figure 2. miR-29 is anti-fibrogenic in C2C12 cells. (A) C2C12 cells were differentiated (DM) for 0, 1, 2 or 4 days, at which times total RNAs were isolated for qRT-PCR measurement of the expressions of Col 1A1, Col 1A2, Col 3A1, a-SMA or VIM as well as MyHC, a-Actin, Troponin and MyoG. Expression folds are shown with respect to 0 hr cells where normalized copy numbers were set to 1. Data are plotted as mean 6 S.D. (B and C) C2C12 cells were transfected with negative control (NC) or miR-29 oligos and differentiated for 48 hrs, at which time total RNAs were isolated for qRT-PCR measurement of the expressions of Myogenin, Troponin, a-Actin or MyHC as well as Col 1A1, Col 1A2, Col 3A1, a-SMA or VIM. Expression folds are shown with respect to NC cells where normalized copy numbers were set to 1. Data are plotted as mean 6 S.D. (D) C2C12 cells were transfected with negative control (Anti-NC) or Anti-miR-29 oligos and differentiated for 48 hrs, at which time total RNAs were isolated for qRT-PCR measurement of the expressions of Col 1A1, Col 1A2, and Col 3A1. Expression folds are shown with respect to Anti-NC cells where normalized copy numbers were set to 1. Data are plotted as mean 6 S.D. (E) Wild type (WT) or Mutant Col 1A1, Col 1A2, or Col 3A1-39UTR luciferase reporter constructs were transfected into C2C12 cells with indicated miRNA or negative control (NC) oligos. Luciferase activities were determined at 48 h post-transfection and normalized to b-Galactosidase protein. Relative luciferase unit (RLU) is shown with respect to NC cells where normalized luciferase values were set to 1. The data represent the average of three independent experiments 6 S.D. (F) A schematic illustration of base pairing between mmu-miR-29c with 3078–3099 region on 39UTR of mouse Lims1. (G) Lims1 protein expression was measured in NC or miR-29 stable C2C12 cells by Western blotting using GAPDH as a loading control. (H) NC or miR-29 stable cells were differentiated and Lims1 expression levels were measured at the indicated time points. (I) C2C12 cells were transfected with negative control (Anti-NC) or Anti-miR-29 oligos and differentiated for 48 hrs, at which time total RNAs were isolated for qRT-PCR measurement of the expressions of Lims1. Expression folds are shown with respect to Anti-NC cells where normalized copy numbers were set to 1. Data are plotted as mean 6 S.D. (J) Wild type (WT) or Mutant Lims1-39UTR luciferase reporter constructs were transfected into C2C12 cells with miR-29 or negative control (NC) oligos. Luciferase activities were determined at 48 h post-transfection and normalized to b-Galactosidase protein. doi:10.1371/journal.pone.0033766.g002 In addition to ECM molecules, many cell adhesion genes are down-regulated in miR-29 expressing cells (Table S4). Among them, Lims1 (also called PINCH) is a five LIM domain protein involved in the regulation of integrin-mediated cell adhesion [24]. Interestingly, Lims1 was predicted to contain miR-29 binding sites in their 39UTR regions (Fig. 2F), indicating that it may be a direct target of miR-29. Indeed, Lims1 protein was evidently down- regulated by over-expression of miR-29 in C2C12 cells (Fig. 2G). The mRNA expression of Lims1 was also down-regulated in miR- 29 expressing cells at all time points of differentiation comparing to NC cells (Fig. 2H). Knock-down of miR-29, on the other hand, led to opposite augmenting effect on Lims1 expression (Fig. 2I). Additionally, activities of the reporter with Lims1 binding site were significantly inhibited by miR-29 expression while mutation of this site abolished the inhibition (Fig. 2J). Together, these data demonstrate that Lims1 is a direct target of miR-29. TGF-b suppresses miR-29 expression during myoblast conversion to myofibroblast Having gained insights into the role of miR-29 during the conversion of myoblasts to myofibroblasts, we now turned our attention to its upstream regulator by asking: what leads to the down-regulation of miR-29 in this process? TGF-b has been individuated as the major inducer of myogenic cell into fibrogenic cells but the underlying mechanism is still largely obscure. We thus speculated that the pro-fibrogenic action of TGF-b mediated through miR-29 represents a novel signaling event contributing to fibrogenic conversion of myoblasts. Subsequently, the effects of TGF-b in myogenic and fibrogenic differentiation of C2C12 cells were evaluated. In agreement with previous finding [10], TGF-b treatment of C2C12 cells led to significant delay of myogenic program whereas the expressions of a number of fibrotic genes were increased (Fig. S3A–E). Further IF staining revealed that TGF-b treatment induced a loss of MyoD whereas the a-SMA is increased. In addition, both cell proliferation rate and cell mobility were increased (Fig. S3F–G). These results indicated a conversion of C2C12 to myofibroblasts. As shown in Figure 3A, very low level of a-SMA was detected in untreated cells where MyoD is highly expressed. However, under TGF-b treatment, a-SMA staining was induced and cells exhibited typical a-SMA filament bundles characteristic of myofibroblasts (Fig. 3A and Fig. S3H). We noticed that the cells with strong a- SMA signal completely lost MyoD (a-SMA+/MyoD2) and also assumed a myofibroblast morphology with large and more spread- out looks (Fig. 3A, cells inside the dashed oval), but those with weak a-SMA signal still maintain MyoD staining (a-SMA+/ MyoD+, Fig. 3A, arrow), probably representing an intermediate stage during the conversion. Together, these data suggest that myogenic and fibrogenic differentiations represent two opposite fates of myoblasts; TGF-b promotes the fibrogenic differentiation of C2C12 while suppressing the myogenic program. This is inverse to the effect of miR-29 (Fig. 2), suggesting that TGF-b may function upstream of miR-29 as a suppressor. Next, the potential inhibitory role of TGF-b on miR-29 was examined. Results demonstrated that TGF-b treatment (+) markedly reduced miR-29 expression (Fig. 3B). Furthermore, it exerted a dose-dependent inhibition on miR-29 promoter activities (Fig. 3C), suggesting that the inhibition could be at the transcriptional level through direct action on miR-29 promoter. Next, we sought to determine whether TGF-b repression is biologically functional in terms of regulating the pro-myogenic and anti-fibrogenic action of miR-29. As expected, miR-29 stable cells (Fig. 3D, Lane 5–8) displayed accelerated myogenic differentiation vs NC cells (Lane 1–4). TGF-b treatment led to an obvious delay in the myogenic program in both NC (Lane 9–12) and miR-29 (Lane 13–16) cells, suggesting that TGF-b acts upstream of miR- 29 in antagonizing its pro-myogenic action. Although the addition of miR-29 oligos rescued the anti-myogenic effect of TGF-b, it is still largely existent. This implicates that other downstream pathways could also mediate the effect of TGF-b. The above Western blotting data were also supported by IF staining of MyHC (Fig. 3E) and RNA analysis of myogenic markers (Fig. S3I). In a similar fashion, we examined the effect of TGF-b on the anti- fibrogenic action of miR-29. Expectedly, TGF-b treatment abrogated the suppression of miR-29 on Collagens and a-SMA as well as Lims1 (Fig. 3F–H). Together, these data support that TGF-b acts upstream of miR-29 to antagonize its pro-myogenic and anti-fibrogenic effect in C2C12. On the other hand, miR-29 partially attenuates both the pro-fibrogenic and anti-myogenic actions of TGF-b. TGF-b repression on miR-29 promoter is transcriptionally mediated by Smad3 Given that Smad proteins transmit most of the transcriptional effect exerted by TGF-b, subsequently we examined their involvement in the down-regulation of miR-29. For this purpose, myoblasts transfected with specific siRNAs, capable of attenuating the expressions of Smad2, Smad3, or Smad7 (Fig. 4A), were tested for the responsiveness to TGF-b in regard to inhibiting miR-29. As shown in Figure 4B–C, knockdown of Smad3 but not Smad2 PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e33766