Figure 7
The negative role of miR-203 in skeletal muscle differentiation is carried out, in part, through the inhibition of MEF2C expression. (a) The expression of MEF2C protein during dwarf chicken leg muscle development at different embryonic days. (b) Expression of MEF2C protein during primary myoblast differentiation. (c) The predicted binding site and mutated site (green) of miR-203 in the 3’-UTR of MEF2C. (d) The predicted binding site 1 of miR-203 in the 3’-UTR of MEF2C is highly conserved among vertebrates. (e) Luciferase reporters were transfected into DF-1 cells with the miR-203 mimic or control duplexes, and luciferase activity was determined 36 h after transfection. (f) After 3 days of transfection, miR-203 significantly repressed MEF2C protein expression at DM3, DM5 and DM7. The mRNA levels of MYOM1, MYOM2 and MCK are all decreased significantly. (g) After 3 days of transfection, anti-miR-203 significantly repressed MEF2C protein expression at DM3. The mRNA levels of MYOM1, MYOM2 and MCK are all increased significantly. (h) Primary myoblasts were transfected with anti-MEF2C siRNA or negative control duplexes at GM, DM2 and DM4, respectively, and then were incubated in differentiation medium for 3 days before qPCR and immunoblotting. (i) Microscopic images of myotubes at DM7 that were transfected with si-MEF2C or negative control duplexes at DM4. (j and k) Cells were fixed and immunostained for desmin after 3 days of si-MEF2C and negative control duplex transfection at DM4, and then were quantified for the desmin+ nuclei presented in cells with the indicated number of nuclei. All of the results are expressed as the mean±S.E.M. of three replicates. *P<0.05; **P<0.01; ***P<0.001
