Fig. 2

miR-1246 downregulates CPEB4 expression by directly targeting its 3’ UTR. a Scheme for the identification of candidate genes combining microarray assays and a target prediction algorithm. b The mRNA expression levels of the predicted genes in A549 and H226 cells expressing miR-1246 or vector were evaluated by real-time PCR. c Dual-luciferase activity assays were used to determine the binding potential between miR-1246 and the 3’ UTR of these candidate genes. Renilla luciferase activity was detected as an internal control. d Western blot assays of the TRAK2 and CPEB4 protein levels in A549, H226 and HEK 293 T cells. e The sequences of the putative miR-1246-binding site in the wild-type and mutant (underlined) CPEB4 3’ UTR. f The putative binding sequences for miR-1246 within the human (Has), chimpanzee (Ptr), rhesus (MMl) and mouse (Mmu) CPEB4 3’ UTR. Seed sequences are indicated as emphasized with shadow. g The relative luciferase activity of luciferase reporters with wild-type or mutant CPEB4 3’ UTRs was determined in HEK 293 T cells, which were co-transfected with the miR-1246 mimic or negative control (nc). Renilla luciferase activity was analyzed as an internal control. There is statistical significance between the wild-type group and the mutant group with miR-1246 transfection. h The protein levels of CPEB4 were determined by western blot assays in H226 cells infected with miR-1246 or mock vector and H226 cells transfected with the miR-1246 inhibitor or negative control (nc). * p < 0.05, ** p < 0.01, *** p < 0.001