Mutation of the putative recognition element abolishes miRNA-361-5p-mediated regulation of a VEGFA 3′-UTR reporter.
(A) Schematic representation of the luciferase reporter constructs, indicating the VEGFA 3′-UTR fragment fused to Renilla luciferase, the predicted miRNA recognition element (MRE) for miR-361-5p, and the firefly luciferase gene used for normalization. (B) Sequence alignment of miR-361-5p with the wild type (wt) VEGFA 3′-UTR and mutated reporter. The seed sequence (bold) and mutated residues (red) are highlighted. (C) HEK293 cells were co-transfected with either of the indicated luciferase reporters and a miR-361-5p mimic, antisense inhibitor, or the respective control. Data were obtained from three independent experiments, performed in triplicate. For each experiment, mean ratios of Renilla and firefly luciferase activities were normalized to those of psiCHECK-2-transfected cells, then to Pre- or Anti-miR control-transfected cells. Mean values of three independent experiments ± S.D. are plotted. Two-tailed, unpaired t-tests were used to calculate P values (single asterisks denote P values<0.05).