Figure 2

Knockdown of miR-133 repressed myoblast differentiation and promoted cell proliferation. (a) In myoblasts and myotubes, miR-133b inhibitor significantly repressed endogenous expression of miR-133. Inhibitor NC and miR-133b inhibitor were transfected into C2C12 cells cultured in six-well plates. Total RNA of transfected cells was isolated from cells cultured in the growth medium (GM) for 24 h or differentiation medium (DM) for 36 h after transfection. Expression of miR-133 was analyzed by qPCR. (b) Expression of myogenin and MyHC-2d was significantly downregulated by miR-133b inhibitor. Total RNA of C2C12 cells transfected with miR-133b inhibitor or inhibitor NC and cultured in the DM for 36 h after transfection was isolated, and the expression of myogenin and MyHC-2d was analyzed by qPCR. (c) Inhibition of miR-133 expression promoted myoblasts’ proliferation. C2C12 cells transfected with miR-133b inhibitor or inhibitor NC were collected for cell cycle analysis approximately 30 h after transfection. Cells were stained by propidium iodide and subjected to fluorescence-activated cell sorting analysis by flow cytometry. (d) Inhibition of miR-133 expression repressed myoblasts differentiation. Inhibitor NC or miR-133b inhibitor was transfected into C2C12 cells cultured in 24-well plates, respectively. After transfection, cells were switched into the GM for 24 h. Then, cells were transferred to the DM for another 4 days before immunostaining for MyHC (green). Nuclei were stained with DAPI. (e) Area of myotubes stained in (d). Myotube area was calculated by Image Pro Plus software. Error bars represent S.D. from three independent experiments (*P<0.05)