combining the effects of both location and size as a new criterion for the prediction of MREs and their cognate miRNAs to FindTar, a computational algorithm developed in our lab, decreased the false positive rates and increased the accuracy of MRE prediction significantly. Results Putative miRNAs for VEGF regulation VEGF is an important regulator of physiological or pathological angiogenesis and is involved in embryogenesis, wound healing, and tumorigenesis [41]. In this study, we used VEGF as a model to investigate the effect of the secondary structure of the non-seed region in the miRNA:MRE duplex on miRNA-mediated gene regulation. Previously, we developed an algorithm, FindTar version 1.0, using the generally recognized criteria to predict the binding interactions of miRNA:MREs and their secondary structures. The cut-offs 224 and 215 kcal/mol were chosen subjectively to avoid predicting too few or too many putative miRNAs targeting VEGF. When strict screening criteria were used, which included a minimal free energy (DG) below 224 kcal/mol, tail scores of miRNA:MREs duplexes above 15, seed windows at nt2–7 with conservation without G-U wobble, 16 MREs for 15 potential miRNAs were predicted as Group 1 (see Table S1A) in the VEGF 39-UTR. When we relaxed the screening criteria to DG between 224 and 215 kcal/mol, tail score above 5, plus elastic seed windows (nt1–6, nt2–7, or nt3–8) with tolerance for a single G-U wobble in the seed region[42], hundreds of MREs and miRNAs were predicted. From these miRNAs, we randomly chose 15 miRNAs as the experimental Group 2 (Table S1B). Hypoxia- induced CNE cells (nasopharyngeal carcinoma cell line) were transfected with synthetic miRNA duplexes, including all the miRNAs in Group 1 and 2. A random sequence (NC), miR-224, mutated miR-16, and miR-20a that did not have MREs in the VEGF 39-UTR were used as negative controls. VEGF expression was measured by ELISA. The experiments indicated that 23 of the 30 miRNAs significantly repressed VEGF expression by over 20%, compared to negative controls (Figure 1). To confirm the repressive action of the miRNAs through forming miRNA:MRE duplexes, we inserted two fragments of the VEGF 39-UTR into a luciferase expression vector, generating the luciferase reporter constructs pRL-VEGF-Con1 and pRL-VEGF- Con2. pRL-VEGF-Con1 contains a fragment located at nt31-216 of the VEGF 39-UTR, while pRL-VEGF-Con2 contains a fragment at nt703-944. Of the 30 miRNAs tested in the ELISA assay, 21 harbored a single MRE in pRL-VEGF-Con1 and/or pRL-VEGF-Con2. These miRNAs were co-transfected with their respective reporters into COS-7 cells and the levels of Renilla luciferase (RL) were measured to determine the repressive effects of these miRNAs. These experiments indicated that most of the selected miRNAs produced a direct effect with significant repression of luciferase activity as compared with the controls (Figure 2). The relationship between the central loops of miRNA:MRE duplexes and the repressive efficiency of miRNAs We analyzed the relationship between the central loop of miRNA-mRNA duplexes and the repressive efficiency of miRNAs in Groups 1 and 2 on VEGF expression, and found that the location of the central loop in miRNA:MRE duplexes appeared to play an important role. The loops of the miRNA:MRE duplexes in Table S1 were divided into three types according to their location in the duplex. Loops starting between nt9 and nt11 inclusive of the MicroRNA Central Loops miRNAs were designated standard loops, before nt9 as Type I decentered loops, and after nt11 as Type II decentered loops (Figure 3A). Thus, of the 30 total miRNA:MREs tested, there were 21 miRNAs with standard central loops, 2 with Type I decentered loops, and 7 with Type II decentered loops. Of the 24 functional miRNAs with a repression ratio of VEGF expression .20%, 19 harbored a standard central loop, and only 5 contained Type II decentered loops. On the other hand, amongst the 7 non- functional miRNAs, 2 had Type II decentered loops, 2 had Type I decentered loops, and only 3 had standard central loops. Our analysis indicates that most of the MREs with a decentered loop were either false positives or highly inefficient. According to these data, we hypothesize that the central loop is one of the important factors that guide miRNA-MRE interactions. However, our data only included interactions between miRNAs and the 39-UTR of VEGF, and many VEGF regulatory miRNAs have more than one MRE in the 39-UTR of VEGF with an overlap of target sites, and there is a degree of similarity between different microRNAs to each other. Therefore, more data was needed to confirm our hypothesis on the importance of the loop in the miRNA:MRE duplexes in miRNA functioning. The published data on miRNA target identification by experiments and bioinformatics approaches, which were available on the website DIANA Tarbase (www.diana.pcbi.upenn.edu/ tarbase.html)[31,32,43–49], support our hypothesis. We used these data to validate the effect of the loop structure of miRNA:MRE duplexes on the repression of gene expression. To avoid the incongruency brought by different standards or outcomes of RNA secondary structure and free energy predicted by distinct algorithms, we re-calculated the secondary structures of miRNA:MRE duplexes and their free energy for all the predicted miRNAs obtain from the online database by RNAcofold, a program for the prediction of hybrid structures of two RNA sequences (www.tbi.univie.ac.at/,ivo/RNA/)[50–52]. Thus, we provided a relatively equal platform or prerequisite to compare the structure of all these microRNA targets. To exclude the interference of multiple MREs of a single miRNA for one gene, we only took those miRNAs that have single MREs on one gene into account, and a total of 20 miRNAs were selected (Table S2). Analysis of the effect of the loop structure of miRNA:MRE duplexes on the repression of gene expression showed that 18 of the 20 microRNAs had their loop starting from p9 to p11, while only 2 had Type I decentered loops. Therefore, while much attention has focused on the seed region in the identification of miRNA targets[53,54], it seems that other factors such as the location of the loops also have a great impact on gene repression. The miRNAs with a single MRE in pRL-VEGF-Con1 and/or pRL-VEGF-Con2 (Table S3) were selected to investigate the relationship between miRNA repression efficiency and the secondary structure of miRNA:MRE duplexes. The central loops are scored as follows: (i) to get a location coefficient according to the locations of loops: 1 for standard loop, 0.75 for type II decentered loop, 0 or 0.5 for type I decentered loops starting before nt7 inclusive or at nt8; (ii) to get the size coefficient according to the sizes of the loops: 10 for 1 bp, 20 for 2 bp, and 25 for 3 bp. If the size of the loop was more then 3 bp, the size coefficient would drop to 20. The total loop scores were calculated by multiplying the location coefficient with the size coefficient. The relationship between miRNA efficiency and the central loop was analyzed using correlation and linear regression. The central loop scores demonstrated a significant correlative relationship with miRNA repressive efficiency (Figure. 3B). The tail score (Figure 3C) and minimum free energy (Figure 3D) did not show significant relationships with gene repression. PLoS ONE | www.plosone.org 2 March 2008 | Volume 3 | Issue 3 | e1719