Co-regulation of miRNAs to angiogenic factors.

<p>CNE cells were induced with or without DFOM (6–1), transfected with <i>miR-15b, miR-16, miR-20a</i>, or <i>miR-20b</i> (6–2) and transfected with Construct I or Construct II (6–3). Cell lysate was collected, and the expression of angiogenic factors uPAR (A), COX2 (B), c-MET (C), and PTN (D) were determined by Western Blotting.</p

Analysis of miRNA binding sites in VEGF.

<p>(A) The map of miRNA binding sites in the VEGF 3′-UTR in accordance with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000116#pone.0000116.s001" target="_blank">Table S1</a>. Three bioinformatics algorithms, RNAhybrid, miRanda, and FindTar, were used to predict miRNA binding sites in the VEGF 3′-UTR. A binding site map of the VEGF 3′-UTR was generated with the putative binding sites predicted by at least two bioinformatics algorithms. Subsequently, two miRNA binding site-condense regions (MBSCR) were uncovered, that is, 160–195nt and 820–860nt MBSCR. (B) A common miRNA binding site located in 160–185nt shared by 12 miRNAs. (C) The miRNAs that share this common miRNA binding site belong to four different miRNA clusters. (D) The seed region of this common miRNA binding site is highly conserved among mammals. (E) RNAcofold was used to analyze the secondary structure of the common miRNA binding site and its flank regions in the VEGF 3′-UTR. The common binding site is located in an unstable region with a multi-branching loop-like RNA structure.</p

Luciferase activity assay.

<p>Luciferase reporter constructs were generated through placing fragments of 185 bp (Construct I, sequence from nt31–216 of the VEGF 3′-UTR) and 241 bp (Construct II, sequence from nt703–944 of the VEGF 3′-UTR) at the 3′ end of luciferase gene in pRL-TK. COS-7 cells were co-transfected with a luciferase reporter vector and an miRNA which has a putative binding site in either Construct I (A) or Construct II (B). Luciferase activity was measured to determine the effects of these miRNAs on luciferase translation. In addition to a random sequence (NC), <i>miR-29, miR-150</i>, and <i>miR-383</i> which have putative binding sites in the 3′-UTR of VEGF but not on Construct I or II, as predicted by all of the algorithms, were employed as negative controls. *, p<0.05; **, p<0.01.</p

VEGF and miRNA expression in CNE cells.

<p>Cell lysate and culture medium from DFOM-induced CNE cells were collected. RT-PCR (A) and ELISA (B) assays were performed to determine VEGF expression at the mRNA and protein levels. CNE cells without DFOM treatment were used as a control. The <i>mir</i>Vana™ miRNA Detection procedure was used to validate miRNA chip data. <i>miR-15b, miR-16, miR-20a</i>, and <i>miR-20b</i> were down-regulated in hypoxia induced CNE cells (C). I: induced, N: non-induced.</p

Effects of different combinations of miRNAs on VEGF expression.

<p>(A) CNE cells were transfected or co-transfected with <i>miR-20a</i> and <i>miR-106b</i>, which share the same binding site, or with <i>miR-20a</i> and <i>miR-361</i>, which target different binding sites. An unrelated random sequence was used as a negative control (NC). A specific sequence targeting VEGF (functioning as siRNA) was used as a positive control (PC). Culture medium was collected 30h after transfection. Repression of VEGF expression was detected by ELISA. (B) miRNA binding site locations of <i>miR-378, miR-125a, miR-93, miR-302d</i>, and <i>miR-373</i> in the VEGF 3′-UTR fragment of Construct II. (C) COS-7 cells were co-transfected with (1) Construct II and <i>miR-378, miR-125a, miR-93, miR-302d</i>, or <i>miR-373</i>; (2) Construct II and <i>miR-378</i> plus either <i>miR-125a, miR-93, miR-302d</i>, or <i>miR-373</i>. Luciferase activity was measured to investigate the effects of different combinations of miRNAs. *, p<0.05; **, p<0.01.</p