Figure 5
c-JUN is a direct target of miR-203 in chickens. (a and b) The mRNA and protein expression of c-JUN in the dwarf chicken embryonic leg muscle and primary myoblast differentiation process. (c) The mRNA and protein expression of c-JUN is significantly reduced after miR-203 transfection in myoblasts. (d) The mRNA and protein expression of c-JUN is significantly increased after anti-miR-203 transfection in myoblasts. (e) The c-JUN 3’-UTR was inserted into the dual-luciferase reporter vector pmirGLO (upper) at the 3' end of the firefly luciferase gene (luc2). Constitutive Renilla luciferase (hRluc-neo) expression was used as an internal normalisation control. The predicted binding site and mutated site (green) of miR-203 in the 3’-UTR of c-JUN is shown (bottom). (f) DF-1 cells were transfected with c-JUN 3’-UTR wild-type and mutant luciferase reporters and co-transfected with miR-203 mimic or control duplexes. The relative luciferase activity was measured 36 h later. (g) Relative p53 and p21 mRNA levels normalised to β-actin mRNA in myoblasts transfected with miR-203 mimic and control duplexes. (h) Relative p53 and p21 mRNA levels normalised to β-actin mRNA in myoblasts transfected with anti-miR-203 and anti-NC. All of the results are expressed as the mean±S.E.M. of three replicates. *P<0.05; **P<0.01; ***P<0.001
