PMC

miR-133 represses IGF-1R at the posttranscriptional level and negatively regulates the IGF-1R/PI3K/Akt signaling pathway, which is involved in skeletal muscle proliferation, differentiation and hypertrophy. Extracellular IGF-1 accelerates miR-133 expression via myogenin induction.

(A) C2C12 cells were transfected with negative control or miR-133 mimics at 50 nM or 100 nM for 36 hours. (B) C2C12 cells were transfected with 50 nM RNA mimics and inhibitors as indicated for 36 hours. (C) C2C12 cells were transfected with 50 nM negative control, miR-133 mimics or anti-IGF-1R siRNA. Proteins were extracted for western blotting against IGF-1R. α-tubulin was served as loading control. The mRNA levels of IGF-1R were determined by semi-quantitative RT-PCR, and GAPDH was used as internal control. (D) C2C12 cells were transfected as in (C). 24 hours after transfection, cells were serum starved for 24 hours and incubated in differentiation medium supplemented with 5 nM IGF-1 for 30 minutes. Proteins were extracted for western blotting against Ser-473 phosphorylated Akt and total Akt. GAPDH was used as a loading control. Representative results from independent experiments (n≥2) are shown. The numbers below the blots represent relative expression levels.

(A) IGF-1R protein and miR-133 levels were determined in hind limb muscles at 18.5 dpc embryos, 2 days postnatal neonates or adults in C57BL/6J mice by western blot or northern blot analysis. GAPDH or U6 snRNA served as loading controls for protein or small RNA. (B) Morphology of proliferating myoblasts maintained in growth medium or differentiated myotubes after serum derivation for 6 days. (C) C2C12 myoblasts were induced to differentiate for up to 8 days. Protein levels were determined by western blotting, miRNA by northern blotting and mRNA by semi-quantitative RT-PCR. Representative results from independent experiments (n≥2) are shown. The numbers below the blots represent relative expression levels. GM represents growth medium and DM represents differentiation medium.

(A) C2C12 cells were induced to differentiate in the presence or absence of 5 nM IGF-1. Morphological change was examined by phase contrast microscopy. (B) miR-133 levels normalized to U6 were determined by quantitative RT-PCR. miR-133 levels in day 1 after differentiation induction were set at 1, and relative expression was shown as fold induction. Data represent the mean ± S.D. of three independent experiments. **p<0.01 vs. day 1 without IGF-1 treatment. Protein extracted from differentiating C2C12 cells in the presence or absence of IGF-1 was used for western blot analysis using a myogenin antibody; α-tubulin served as loading control. (C) C2C12 cells were transfected with myogenin siRNA for 24 hours before induced to differentiate in the presence or absence of 5 nM IGF-1 for another 48 hours. Morphological change was shown. (D) Relative miR-133 levels were determined by quantitative RT-PCR. miR-133 levels in negative control transfectants without IGF-1 treatment were set at 1, and relative expression was shown. Data represent mean ± S.D. of three independent experiments. *p<0.05, **p<0.01 vs. negative control transfectants without IGF-1 treatment. Western blot analysis determined the expression of myogenin protein. Representative results from independent experiments (n≥2) are shown. The numbers below the blots represent relative expression levels.

(A) Seed-matched sequences in the IGF-1R 3′UTRs are in red and conserved regions between aligned sequences are indicated by stars. (B) Schematic representation of luciferase reporter constructs. miRNA-mRNA hybridization structures and folding energies were predicted by RNAhybrid. (C) HEK 293T cells were transfected with psiCHECK2 luciferase reporter vectors containing wild type or mutated miR-133 binding sites downstream of the Renilla luciferase gene (50 ng), and the internal Firefly luciferase gene was used to normalize for transfection efficiency. A pcDNA6.2-miR-133 expression vector or pcDNA6.2-negative control vector was cotransfected (150 ng). Dual-luciferase assays were performed 48 hours after transfection. Normalized luciferase activities of miR-133 transfectants were shown as the percentage relative to pcDNA6.2 transfectant, which was set at 1. Data represent the mean ± standard deviation (S.D.) of three independent experiments. ***p<0.001 vs. pcDNA6.2 transfectants.