miR-27a/b targets and represses <i>Mstn</i> expression.

<p>(A) <i>In silico</i> analysis, using TargetScan algorithms, showing a 8 mer seed match (grey box) between murine miR-27a (mmu-miR-27a) and miR-27b (mmu-miR-27b) and the miR-27a/b binding site located within the <i>Mstn</i> 3′ UTR sequence (mmu-<i>Mstn</i> 3′UTR). (B) Assessment of pMIR-REPORT™ luciferase activity in C2C12 myoblasts co-transfected with the <i>Mstn</i> 3′UTR reporter construct (<i>Mstn</i> 3′UTR) and either control (pcDNA-miR-neg), miR-27a over expression construct (pcDNA-miR-27a), negative control AntagomiR (AntagomiR Neg) or a miR-27a-specific AntagomiR (AntagomiR-27a) for 48h. (C) Assessment of pMIR-REPORT™ luciferase activity in C2C12 myoblasts co-transfected with the mutant <i>Mstn</i> 3′UTR reporter construct (<i>Mstn</i> 3′UTR-mut), where the miR-27a/b binding site has been mutated, and either control (pcDNA-miR-neg), miR-27a over expression construct (pcDNA-miR-27a), negative control AntagomiR (AntagomiR Neg) or a miR-27a-specific AntagomiR (AntagomiR-27a) for 48h. For all pMIR-REPORT™ transfections, luciferase activity was normalized to Renilla luciferase and expressed as fold change relative to control (pcDNA-miR-neg). Bars represent mean values ± S.E.M (n = 3). <i>p</i><0.05 (*) and <i>p</i><0.001(***). qPCR analysis of <i>Mstn</i> mRNA expression (D) and precursor-miR-27a/b (pre-miR-27a/b) expression (E) in Heart, Liver, <i>M. Biceps femoris</i> muscle (BF) and <i>M. Soleus muscle</i> (Sol.) collected from 4-week-old wild type (WT) mice. Bars represent fold change (relative to Heart) ± S.E.M (n = 3) normalized to <i>GAPDH</i> (D) or U6 (E) expression. <i>p</i><0.05 (*) and <i>p</i><0.001(***). qPCR analysis of (F) <i>Mstn</i> and (G) miR-27b expression in C2C12 myoblast cultures differentiated across a time course (24 h, 48 h, 72 h and 96 h differentiation). Bars represent fold change (relative to 24 h control) ± S.E.M (n = 3) normalized to either <i>GAPDH</i> (F) or U6 (G) expression. <i>p</i><0.001(***).