Negative Auto-Regulation of Myostatin Expression is Mediated by Smad3 and MicroRNA-27

<div><p>Growth factors, such as myostatin (Mstn), play an important role in regulating post-natal myogenesis. In fact, loss of Mstn has been shown to result in increased post-natal muscle growth through enhanced satellite cell functionality; while elevated levels of Mstn result in dramatic skeletal muscle wasting through a mechanism involving reduced protein synthesis and increased ubiquitin-mediated protein degradation. Here we show that miR-27a/b plays an important role in feed back auto-regulation of Mstn and thus regulation of post-natal myogenesis. Sequence analysis of <i>Mstn</i> 3′ UTR showed a single highly conserved miR-27a/b binding site and increased expression of miR-27a/b was correlated with decreased expression of <i>Mstn</i> and vice versa both <i>in vitro</i> and in mice <i>in vivo</i>. Moreover, we also show that <i>Mstn</i> gene expression was regulated by miR-27a/b. Treatment with miR-27a/b-specific AntagomiRs resulted in increased <i>Mstn</i> expression, reduced myoblast proliferation, impaired satellite cell activation and induction of skeletal muscle atrophy that was rescued upon either blockade of, or complete absence of, Mstn. Consistent with this, miR-27a over expression resulted in reduced <i>Mstn</i> expression, skeletal muscle hypertrophy and an increase in the number of activated satellite cells, all features consistent with impaired Mstn function. Loss of <i>Smad3</i> was associated with increased levels of Mstn, concomitant with decreased miR-27a/b expression, which is consistent with impaired satellite cell function and muscular atrophy previously reported in <i>Smad3</i>-null mice. Interestingly, treatment with Mstn resulted in increased miR-27a/b expression, which was shown to be dependent on the activity of Smad3. These data highlight a novel auto-regulatory mechanism in which Mstn, via Smad3 signaling, regulates miR-27a/b and in turn its own expression. In support, Mstn-mediated inhibition of <i>Mstn</i> 3′ UTR reporter activity was reversed upon miR-27a/b-specific AntagomiR transfection. Therefore, miR-27a/b, through negatively regulating <i>Mstn</i>, plays a role in promoting satellite cell activation, myoblast proliferation and preventing muscle wasting.</p>