Figure 3.

Nrp-2 is a target for miR-188. A, Left, the predicted hybridization of miRNAs (green) and the Nrp-2 (red) transcript using the RNAhybrid algorithm. The minimum free energy required for the hybridization is indicated. Right, a schematic diagram of rat Nrp-2 mRNA containing the predicted conserved target site of miR-188. B, HEK 293 cells were cotransfected with one of the following combinations using Lipofectamine 2000 (Invitrogen): (1) wild-type 3′-UTR of Nrp-2 in pMIR-REPORT (wt 3′UTR of Nrp-2) alone; (2) wt 3′-UTR of Nrp-2 plus 40 nm miR-188 oligonucleotide; (3) wt 3′-UTR of Nrp-2 plus 80 nm miR-188 oligonucleotide; (4) wt 3′-UTR of Nrp-2 plus miR-SC oligonucleotide; (5) mutant type 3′-UTR of Nrp-2 in pMIR-REPORT (mt 3′-UTR of Nrp-2) alone; (6) mt 3′-UTR of Nrp-2 plus 40 nm miR-188 oligonucleotide; (7) mt 3′UTR of Nrp-2 plus 80 nm miR-188 oligonucleotide; or (8) mt 3′-UTR of Nrp-2 plus miR-SC oligonucleotide. Luciferase activity assays were performed 48 h after transfection using the Luciferase Assay kit (Promega) and were measured with a Centro LB960 reader (Berthold Technologies). The β-galactosidase activity was measured to normalize the luciferase activity. The relative luciferase activity was reduced in the cells cotransfected with the wt 3′-UTR of Nrp-2 plus miR-188 (76.2822 ± 1.2642%, n = 6, **p = 0.003579), compared with the cells transfected only with the wt 3′-UTR of Nrp-2. However, in the cells cotransfected with wt 3′-UTR of Nrp-2 plus miR-SC, no significant difference was observed compared with the cells transfected only with the wt 3′-UTR of Nrp-2. The statistical analysis was performed using a nonparametric Mann–Whitney for wt 3′-UTR of Nrp-2 and independent t test for mt 3′-UTR of Nrp-2, respectively; the data are represented by the mean ± SEM, respectively.