MicroRNA Central Loops Figure 1. Validation of VEGF regulation by putative miRNAs. The effect of putative VEGF-regulative miRNAs on VEGF expression was tested in hypoxia-induced CNE cells by transfecting the cells with siRNA duplexes homologous in sequence to the miRNAs in group 1 (A) and 2 (B). VEGF expression was determined by ELISA. MiRNAs in group 1 were selected using a strict criteria: free energy less than 224 kcal/mol, nt2–7 perfectly pairing with the 59-end of miRNAs, and sequence conservation of target sites across five vertebrate species. MiRNAs in group 2 were chosen from miRNAs that only met relatively relaxed criteria: free energy between 224 kcal/mol and 215 kcal/mol, and an elastic seed window tolerating one G:U wobble in successive 6-mers. Repressive ratio = (1–ELISA value of miRNA/ELISA value of blank)6100%. The blank is the sample from cells without transfection, providing a control for protein expression in the absence of regulation. A random sequence (NC), miR-224, mutated miR-16 (miR-16-M), and miR-20a (miR-20a-M) that do not have MREs in the VEGF 39-UTR were used as negative controls. doi:10.1371/journal.pone.0001719.g001 Effect of loop location on miRNA action Although the results of co-transfection of miRNAs and the reporter pRL-VEGF-Con1 and/or pRL-VEGF-Con2 suggested a tight connection between the loop structure and the repressive effect of miRNAs, direct evidence was still needed to confirm our hypothesis. To do so, we performed site mutation assays (Figure 4). Mutations of miR-17-5p (Figure 4A) and miR-372 (Figure 4B) moved the central loops of the miRNA:MRE duplexes to Type I and Type II decentered loops while keeping the minimum free energy of the miRNA:MREs at similar levels, and the mutation of MREs of miR- 17-5p and miR-372 in pRL-VEGF-Con1 and pRL-VEGF-Con2 also formed Type I and Type II decentered loops (Figure 4D and E). COS-7 cells were then transfected with wild type or mutated miRNAs with different report vectors. The levels of luciferase activity were measured to determine the repressive effects of different miRNA:MRE duplexes. Duplexes with type I or II decentered loops produced significantly lower repressive effects, compared with the miRNA:MRE duplexes with standard loops (Figure 4C and F). PLoS ONE | www.plosone.org 3 March 2008 | Volume 3 | Issue 3 | e1719