Fig. 5.

Multi-miRNAs target JNK/MAPK signaling. a, b Luciferase assays show the effects of multi-miRNAs on reporter constructs containing the wild-type (a) and mutant (b) target fragments of each candidate target gene. MT mutant, WT wild-type. The target sites constructs were co-transfected into C2C12 cells with either the corresponding miRNA or a miR-neg expression vector. The pcDNA6.2-miR-negplasmid (miR-neg) was used as a negative control. The relative luciferase activities are the ratio of renilla/firefly luciferase normalized to the negative control for each target group. c The relative expression of mRNA of the six JNK/MAPK signaling key factors (MEKK1, MEKK2, MKK7, MKK4, JNK1/2, and JUN) in C2C12 cells transfected with mimics of mamiRs or with a miRNA NC by qRT-PCR. d Western blotting showed the effect of the overexpression of mamiRs (1a, 133a, 133b, 206, 128, and 351) on the protein expression of targets of JNK/MAPK signaling (MEKK1, MEKK2, MKK7, and c-Jun). C2C12 cells were transfected with mamiR mimics or with NC. e Relative expression in d were calculated. GAPDH was the internal control. Values are means ± SEM. The statistical significance of difference between two means was calculated with the t-test, *p < 0.05; **p < 0.01