To investigate the role of microRNA (miRNA) in muscle atrophy, we performed microarray analysis of miRNA expression in skeletal muscles of Sham, orchiectomized (ORX) mice, and ORX mice treated with androgen and identified that the expression of miR-23b-3p in ORX mice was significantly higher than that in Sham mice (P = 0.007); however, miR-23b-3p expression in ORX mice treated with androgen was lower (P = 0.001). We also investigated the mechanism by which overexpression or knockdown of miR-23b-3p influences the expression of myosin heavy chain, muscle protein synthesis, ATP activity, and glucose uptake in C2C12 myotube cells. Moreover, we examined the serum miR-23b-3p levels among male subjects with type 2 diabetes and whether the serum miR-23b-3p levels could be a biomarker for muscle atrophy. The overexpression of miR-23b-3p in C2C12 myotube cells significantly upregulated the expression of myosin heavy chain, protein synthesis, ATP activity, and glucose uptake. Reporter assays raised a possible direct post-transcriptional regulation involving miR-23b-3p and the 3′-UTR of PTEN mRNA. Among subjects with type 2 diabetes, serum miR-23b-3p levels in the subjects with decreased muscle mass were significantly higher compared to the levels in the subjects without. Our results indicate that miR-23b-3p downregulates the expression of PTEN in myotube cells and induces the growth of myosin heavy chain. In addition, the serum level of miR-23b-3p can be used as a diagnostic marker for muscle atrophy.
Supplementary Figure 1 Putative recognition of miRNAs in the 3’-untranslated region of phosphatase and tensin homolog (PTEN) mRNA. A: Putative human PTEN-targeting miRNAs and their recognition sites were predicted using TargetScan Human 6.2. miRNA families conserved only in mammals are shown. The putative recognition sites of miR-23 are boxed. B: Sequence-matching between miRNA and the human PTEN 3’-untranslated region at each site is shown.
Supplementary Figure 2 (A)Heat map of the top 20 micro RNAs of the most varied in OCX mice compared to those in sham mice (n=3 each). (B) The ratio of the weight of soleus muscle to body weight (n=6) are shown. (C) The ratio of the weight of plantaris muscle to body weight (n=6) are shown. (D) The relative expression of miR-23b-3p in soleus muscle normalized to U6 (n=6). (E) The relative expression of miR-23b-3p in plantaris muscle normalized to U6 (n=6). (F) The relative expression of mRNAs of indicated genes in soleus muscle normalized to hprt (n=6). Data are mean ± SEM; *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA.