Fig. 2. MiR-15b and miR-322 directly targeted the 3′-UTR of SETD3 gene in vitro.

a Sequence alignment of the short 3′-UTR fragment of the wild-type or the mutant SETD3 was shown. The mutated sites in the seed region of the miRNAs were pointed out by “*“. b, c Luciferase reporter assays were performed using miR-15b (b) or miR-322 (c) co-transfected with a dual-luciferase reporter construct containing different lengths of 3′-UTR of SETD3. WT wild- type, x1 a single 3′-UTR sequence, x3 triple 3′-UTR repeat sequences. d The luciferase reporter constructs containing different 3′-UTR fragments of SETD3 were transfected into C2C12 cells stably expressing pri-miR-15b or pri-miR-322 construct. Luciferase activities from the indicated cells were measured. FL the full length of the 3′-UTR fragment (756 bp). e Relative expression levels of miR-15b and miR-322 shown in d were measured by RT-qPCR. f Luciferase reporter assays were performed using the indicated miRNA inhibitors co-transfected with a dual-luciferase reporter construct containing short length of 3′-UTR of SETD3. The relative luciferase activities were quantified from three biological repeats