Functional analysis of the PKCα 3′ UTR binding sites for the temperature-sensitive miRNAs. (A) The putative binding sequences for hsa-miR-27b-5p, hsa-miR-1260a, and hsa-miR-92a-1-5p in PKCα 3′ UTR are shown. (B) Sequence of pmirGLO-PKCα-WT or pmirGLO-PKCα-Mut generated by cloning repeats of the putative miR binding sites or mutated sequences, respectively, into pmirGLO. The mutated nucleotides in each miR target sequence is indicated by black text for pmirGLO-PKCα-Mut. (C) HEK 293T cells were transfected with a combination of synthetic miRNA mimics for hsa-miR-27b-5p, hsa-miR-1260a, and hsa-miR-92a-1-5p or an equal concentration of non-targeting scrambled control mimic and pmirGLO-PKCα-WT (wild-type) or pmirGLO-PKCα-Mut (mutant), incubated at 37°C for 24 h, and dual luciferase assays were performed. Mean ± SEM. (*) P < 0.05, n = 4.
