Mutation of the putative recognition element abolishes miRNA-361-5p-mediated regulation of a VEGFA 3′-UTR reporter.

<p>(A) Schematic representation of the luciferase reporter constructs, indicating the VEGFA 3′-UTR fragment fused to <i>Renilla</i> 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 <i>Renilla</i> 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 <i>t</i>-tests were used to calculate P values (single asterisks denote P values<0.05).