Figure 4

Target site search for miR-466 in the Prox1 3' UTR. (A) Illustration showing (i) the location of possible seed match sites between miR-466 and the 3' UTR regions and (ii) the sites altered to produce mutant forms of psiC-Prox1. Site-directed mutagenesis was performed to produce mutant versions of the 3' UTR of Prox1 seed match sequence (psiC-Prox1-m1, psiC-Prox1-m2, and psiC-Prox1-m1m2). (B) Seed matches between miR-466 and the mutated 3' UTRs of Prox1. wt, wild type. (C) Luciferase activity was measured in HEK293T cells co-transfected with the miR-466 mimic and a luciferase reporter vector containing the wild-type or mutated 3' UTRs of Prox1. The scrambled control and miR-466 m (mutant form) were used to confirm sequence-specific binding between miR-466 and the 3' UTRs. Luciferase activity was normalized using firefly luciferase activity and expressed as a ratio of the luciferase activity to the activity obtained from the scrambled control-transfected cells. Error bars indicate SDs (n = 3 per experiment). *P < 0.05. †P < 0.01.