Predicted miR-128a targets related to insulin signaling. (A) Alignment of the predicted binding sequence of miR-128a given below the binding site in the 3′ UTR of insulin signaling pathway-related genes, and the mutated 3′ UTR binding sequence below. The miR-128a seed match region is highlighted in bold and mutated region is underlined. (B) Luciferase reporter gene linked to the 3′ UTR of the individual genes (WT) or the 3′ UTR with a mutated miR-128a binding site (Mut) was co-transfected with miR-128a (black bar) or a scrambled miRNA control (CON) expression plasmid (white bar) into 293T cells. Forty-eight hours after transfection, luciferase assay was performed. The transfection of miR-128a overexpression vector significantly inhibited the levels of luciferase activity in cells that were transfected with 3′ UTR-pGL2 construct of individual genes, whereas it had no effect when cells were transfected with mutated 3′ UTR-pGL2 and miR-128a.

miR-128a negatively regulates myoblast proliferation through targeting of IRS1. (A) Myoblasts, where miR-128a is overexpressed or inhibited, were seeded at the same number (10,000 cells per well in a 48-well culture plate) and then counted every 2 days for 8 days. Overexpression of miR-128a repressed myoblast proliferation compared with scrambled control (CON), whereas inhibition of miR-128 promoted proliferation of myoblasts, indicating that miR-128a is the regulator of cell proliferation in myoblasts. Values are represented as mean±s.e. (n = 3). (B) Immunostaining with phospho-histone-H3 antibody (H3-P), which is a mitosis marker. Myoblasts cultured in growth medium were stained with H3-P antibody and the number of H3-P-positive cells was counted. The ratio of H3-P-positive cells to total nuclei was significantly lower in myoblasts where miR-128a is overexpressed than in control myoblasts, whereas anti miR-128-containing myoblasts had more H3-P-positive cells in comparison with control or miR-128-overexpressed myoblasts. This would indicate that myoblast proliferation is regulated by miR-128a expression levels. Values are represented as mean±s.e. (n = 4). Scale bar: 50 µm. (C,D) Protein levels in genes related to insulin signaling during myoblast proliferation indicated that miR-128a induced the decline of IRS1 and phospho-Akt levels, and the elevation of p21 protein levels. All quantitative results are normalized by GAPDH, and are shown as mean±s.e. from three independent experiments. (E) Effect of miR-128a on the expression of myogenic transcriptional factors (Pax7, Myf5, MyoD and Myogenin) during myoblast proliferation. The levels of myogenic factors were slightly elevated by expression of the anti miR-128, although not significantly. These transcription factors were not affected by miR-128a overexpression in primary myoblasts. The expression values were normalized with 18s rRNA expression, and are represented as mean±s.e. (n = 3).

miR-128a does not affect myoblast differentiation, but inhibition of miR-128a promotes myotube hypertrophy. (A) Primary myoblasts where miR-128a is overexpressed or inhibited were induced to differentiate with DMEM medium containing 5% horse serum for 3 days. The differentiated cells were stained with myosin heavy chain (MyHC) antibody and DAPI. The ratio of MyHC-positive cells to total nuclei (DAPI-positive cells) was quantified. There were no changes among each group during myoblast differentiation. Values are represented as mean±s.e. (n = 3). (B) Quantification of myotube formation. Three days after differentiation, the number of nuclei per myotube was calculated and it was found that overexpression of miR-128a induced smaller myotubes with fewer nuclei relative to anti miR-128a-transduced myotubes. Values are represented as mean±s.e. (n = 3). (C) Evaluation of the mRNA levels of myogenic transcriptional factors (Myf5, Pax7, MyoD and Myogenin). Five days after differentiation, total RNA was extracted from myotubes, and the RNAs were subjected to analysis with qPCR. The expression of Pax7 and MyoD in myotubes where miR-128 was suppressed was significantly reduced compared with scrambled control (CON) myotubes. The expression values were normalized with 18s rRNA expression, and are represented as mean±s.e. (n = 3). (D,E) C2C12 myoblasts transduced by anti miR-128 or scrambled miR control lentivirus vectors were induced to differentiate with DMEM medium containing 5% horse serum for 1 and 3 days, and starved with serum-free medium 24 hours before extraction of protein. The protein levels were explored by western blot analysis. Protein levels of IRS1 and Akt phosphorylation were higher in anti miR-128-transduced myotubes compared with that of control myotubes 1 day after differentiation. Three days after differentiation, the level of IRS1 was still higher in anti miR-128 myotubes than in control myotubes, although phosphorylated Akt level was slightly lower in anti miR-128 myotubes relative to that of control myotubes. All quantitative results were normalized by GAPDH, and are shown as mean±s.e. from four independent experiments. (F) C2C12 myoblasts transduced by anti miR-128 or scrambled control lentivirus vectors were induced to differentiate for 5 days, and starved with serum-free medium 24 hours before extraction of protein. The protein levels of IRS1, INSR, PIK3r1, and Akt phosphorylation were analyzed and quantified, indicating that IRS1 and phospho-Akt levels were reduced whereas INSR and PIK3r1 levels were not altered in anti miR-128-expressing myotubes. Quantitative data were normalized by GAPDH, and are shown as mean±s.e. (n = 3). (G) Inhibition of miR-128 induced myotube hypertrophy. C2C12 myoblasts transduced by anti miR-128 or scrambled control lentivirus vectors were induced to differentiate for 5 days, and then stained with myosin heavy chain (MyHC) antibody (red) and DAPI (blue). Myofiber diameter was measured and quantified. Representative data show that anti miR-128-expressing myotubes were thicker than control-expressing myotubes. Values are shown as mean±s.e. Scale bar: 50 µm.

Inhibition of miR-128 with minicircle vector increases skeletal muscle mass and fiber size. (A) C57BL/6 wild type mice were intravenously injected with 50 µg of minicircle vectors, which have the H1 promoter with anti miR-128 or scrambled miRNA, every 4 days, and each animal was analyzed for body weight. The results indicated that the body weight of anti miR-128-transduced mice was heavier than control (CON) mice. Values are shown as mean±s.e. (n = 4). (B) Thirty-five days after first injection, muscles from mice where anti miR-128 or scrambled miRNA was transduced via minicircle were isolated and each muscle was weighed. The results indicate that the volume of muscles was increased in anti miR-128-transduced mice in comparison with control mice. Values are represented as mean±s.e. (n = 3). (C) The expression levels of miR-128a target genes in the GA muscle from mice where anti miR-128 or scrambled miRNA was transduced were investigated by qPCR. The expression levels of miR-128a targets were elevated in anti miR-128-transduced muscle relative to control muscle, indicating that the expression of miR-128a targets was effectively promoted by anti miR-128 minicircle vector injection in vivo. Values are shown as mean±s.e. (n = 4). (D) QC muscles were isolated 35 days after first injection, sectioned and then stained with H&E. Images show representative H&E staining of QC muscle from mice where anti miR-128 (right) or scrambled miRNA (left) was transduced. The graph shows the frequency distribution of muscle fiber diameter, revealing that transduction of anti miR-128 via minicircles promoted muscle hypertrophy in QC muscles. Values are shown as mean±s.e. (n = 3). Scale bar: 100 µm. (E) Expression levels of Myh7 (MyHC type I slow), Myh2 (MyHC type IIa), Myh1 (MyHC type IIx) and Myh4 (MyHC type IIb) in GA muscles were quantified by qPCR, revealing that there were no differences between anti miR-128- or scrambled miRNA-transduced mice. The expression values were normalized with 18s rRNA expression, and are represented as mean±s.e. (n = 3).