miRNAs associated with translating mRNAs, and a specific miRNA-targeted mRNA present in polysomes [61–63]. An miRNA:mRNA duplex with a flexible loop would have the ability to form different conformations to meet all of the needs in the complex process of translational repression. Materials and Methods MicroRNA and mRNA 3-9UTR Database Human microRNA sequences (version 8.2) were retrieved from the miRBase website[64] (http://microrna.sanger.ac.uk/sequences/ index.shtml). Sequences of the 39-untranslated region (39-UTR) of VEGF were retrieved from human (Homo sapiens), mouse (Mus musculus), rat (Rattus norvegicus), chimpanzee (Pan troglodytes), and Cow (Bos taurus) using the Ensembl Database (http://www.ensembl.org). Algorithm of FindTar Version1.0 We defined a relatively relaxed criterion for the seed region of the MRE so that more possible miRNA targets could be included in the primary screening. A flexible miRNA ‘‘seed window’’ (nt1– 6, 2–7 and 3–8 counted from the 59 end of the miRNA) was used to scan the 39-UTRs of a gene in the order of 59-39 for potential target sites. In the seed windows, we searched for a perfectly Watson-Crick–base-paired stretch of 6 nt but tolerated one G-U wobble. After the seed regions were identified, the upstream flanking region of the seed and the seed region itself was extracted for Dynamic programming. The miRNA:mRNA duplex and the MRE sequences of the VEGF 39-UTR were identified with Dynamic hybridization. The distal sequences in the miR- NA:mRNA duplex were scored with revised Gotoh’s and Marks’ methods [37,65]. A score of +5 was given to G:C and A:T pairs, +2 to G:U wobble pairs, and 23 to mismatch pairs, with the gap- open and gap elongation parameters set to 28.0 and 22.0 respectively. The final scores of the miRNA:mRNA duplexes were the sum of single residue-pair match scores [37]. Then the sequences of MREs identified by Dynamic hybridization in the VEGF 39-UTR and potential miRNA were used as input in RNAcofold to predict the RNA secondary structures of the miRNA:mRNA duplexes. If the length of the MRE identified by Dynamic programming was not long enough to form the miRNA:mRNA duplexes calculated by RNAcofold, the duplex would be simulated again using the same mRNA sequence. This time, the sequence used would begin further upstream, such that the region used was 40nt in length including the seed region. RNAcofold most lately released in the Vienna RNA Package 1.5 beta version (www.tbi.univie.ac.at/,ivo/RNA/) was incorpo- rated into the FindTar version 1.0 algorithm. Sequences of MREs in the VEGF 39-UTR and potential miRNA were input to precisely predict the RNA secondary structures of the miR- NA:mRNA duplexes by calculating the minimum free energy (DG) of the whole miRNA:mRNA duplex. Algorithm of FindTar Version2.0 In order to decrease the number of false positives in the prediction of putative miRNA targets, the central loop law was introduced into the FindTar version 1.0 to produce FindTar Version2.0 (http://bio.sz.tsinghua.edu.cn/findtar/). There were three elements in the central loop law: loop position, loop size, and loop priority. (1) Loop position coefficient: 0 for loops starting before nt7 inclusive, 0.5 for loops starting at nt8, both named Type I decentered loops; 1 for loops starting between nt9 and nt11 inclusive, named the standard loop; 0.75 for loops starting after nt12 inclusive, named the Type II decentered loop. (2) Loop size MicroRNA Central Loops coefficient: 10 for loops with a size of 1 bp, 20 for 2 bp, 25 for 3 bp. When the loop size is above 4 bp inclusive, the coefficient is back to 20. (3) Loop priority law: this criterion was defined to avoid the ‘‘noise’’ bulge which may confuse the central loop scoring system. In order to make it clear which loop should be scored and which should not, the priority for scoring a loop was defined as the first loop which met the following criteria: (a) the first loop starting before nt9 of more than 1 bp; (b) the first loop starting between nt9 and nt11 of any size–if there is more than one loop, then the longest loop will be chosen; (c) the first loop starting after nt11 of any size. Finally, the total loop score was calculated by multiplying the location coefficient with the size coefficient. The central loop score was applied as a filter after all the miRNA:MREs were predicted. All these duplexes were scored, with a higher score equivalent to a higher probability of functioning. Preparation of Reporter Vectors The construction of luciferase report vectors, named pRL- VEGF-Con1 and pRL-VEGF-Con2, has been described previous- ly[40]. Briefly, two fragments of the VEGF 39-UTR with multiple MREs were PCR-amplified using primers with the following sequences: construct 1 sense, 59-cgttctagagtttcgggaaccagatctc; antisense, 59-aacactagtaatgcttccgccggagt; construct 2 sense, 59- tcttctagacaggtcagacggacag; antisense, 59-acaactagtctcttctcttcgccgg. Two copies of each fragment were cloned downstream of the stop codon in pRL-TK (Promega Corp., Madison, WI, USA). pRL- VEGF-Con1 contains nucleotides nt31-216 of the VEGF 39-UTR, while pRL-VEGF-Con2 contains nucleotides nt703-944 of the VEGF 39-UTR. The site-specific mutants of pRL-VEGF-Con1 and pRL-VEGF-Con2 were also established by using Takara MutanB- EST Kit (Takara Corp., Dalian, PRC) according the method provided by the manual. Cell Culture and Hypoxia Induction COS-7 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (GIBCO, Carlsbad, CA, USA) containing 10% fetal bovine serum (GIBCO, Carlsbad, CA, USA), and the CNE cells obtained from Kunming Cell Bank (Kunming, China) were cultured in RPMI 1640 (GIBCO, Carlsbad, CA, USA) containing 10% fetal bovine serum. Hypoxia induction was performed by adding desferrioxamine mesylate (DFOM) (Sigma- Aldrich Co., Saint Louis, MO, USA) in the culture medium to a final concentration of 130 mM. Preparation of an siRNA duplex homologous to the miRNA sequence, transfection and dual luciferase assay SiRNA duplex homologous to the miRNA sequences were synthesized and purified by Shanghai GenePharma Co. Ltd (Shanghai, China). pRL-TK plasmids bearing MREs in the 39- UTR (400 ng for CNE, 100 ng for COS-7) were co-transfected with the pGL-3 reporter plasmid (100 ng for CNE, 5 ng for COS- 7) and 20 pmol of siRNA/miRNAs into CNE or COS-7 cells (,36104, 24-well cell culture plates) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in accordance with the manufacturer’s instructions. 26–32 hours after transfection, luciferase activity was determined using the Dual Luciferase Reporter Assay System (Promega Corp., Madison, WI, USA). ELISA assay The supernatant harvested from cell culture 30 hrs after transfection was assayed using human VEGF ELISA kits from R&D Systems (Minneapolis, MN, USA) and read by GENios PLoS ONE | www.plosone.org 10 March 2008 | Volume 3 | Issue 3 | e1719