miR-146a-5p acts as a negative regulator of TGF-β signaling in skeletal muscle after acute contusion

Growing evidence suggests the importance of microRNAs (miRNAs) in stress signaling pathways. Transforming growth factor-beta (TGF-beta) is a potent cytokine that promotes the development of skeletal muscle fibrosis after acute contusion. However, how miRNAs are involved in TGF-beta signaling and confer the robustness of TGF-beta-induced fibrotic response remains to be fully elucidated. Here, we demonstrated that miR-146a-5p (miR-146) levels were reduced in a fibrotic mouse model after acute muscle contusion. It was also found that TGF-beta treatment decreased the expression of miR-146 in vitro in a dose-and time-dependent manner. In addition, overexpression of Smad3 and Samd4, two key players in TGF-beta signaling, suppressed the expression of miR-146 in muscle cells. Overexpression of miR-146 inhibited the expressions of fibrosis markers both in vitro and in vivo. Moreover, increase in the expression of miR-146 in muscle cells was able to attenuate the effect of TGF-beta on the expressions of fibrosis markers. Mechanistic analysis revealed that Smad4 is a direct target of miR-146 in muscle cells. Furthermore, the anti-fibrotic effect of miR-146 could be blocked by overexpression of Smad4 in vivo. These results suggest that Smad4 is down-regulated by miR-146 in skeletal muscle. Taken together, our results indicate that the anti-fibrotic miR-146 is a component of TGF-beta signaling. It is down-regulated by Smad protein, and can inhibit the expression of Smad4. Our study suggests that miR-146 might have a therapeutic potential to reduce skeletal muscle fibrosis after injury.