predominant tumor angiogenesis factor in the majority of human cancers, including those of the breast, colon, lung and prostate [7,8]. Invasive SCC also expresses increased levels of VEGFA, particularly in the leading front of the tumor, which is an intuitive site for the induction of angiogenesis [9]. Furthermore, over- expression of (murine) VEGFA resulted in enhanced invasion and angiogenesis in experimental human SCC [10]. In some other cancers, such as head and neck squamous cell carcinoma, increased expression of VEGF has been associated with pro- gression to a more aggressive phenotype, both clinically and in experimental systems [11]. Similarly, increased VEGFA expres- sion correlates with greater metastatic potential of melanoma, and its expression is high in melanoma metastases themselves [12,13]. In the skin, VEGFA is mainly secreted by epidermal keratinocytes. Its expression is up-regulated in response to hypoxia [14], activation of epidermal growth factor (EGF) receptor signaling via EGF or transforming growth factor (TGF)-a, and to a number of cytokines including tumor necrosis factor (TNF)-a, TGF-b, fibroblast growth factor-7 and others [15,16]. Interestingly, it was shown that heterozygous deletion of the VEGFA 39-untranslated region (39-UTR) in mice leads to a two- to three-fold increase in VEGFA levels and embryonic lethality following cardiac failure, thus suggesting the presence of important regulatory elements in its downstream untranslated region [17]. Indeed, VEGFA expression appears to be excessively regulated at the post-transcriptional level. For example, at least twelve protein isoforms have been described, resulting from alternative splicing, as well as the use of alternative promoters or ribosome entry sites [18]. Moreover, its 39-UTR contains several putative or verified stabilization/destabilization elements and alternative polyadenyla- tion signals, which may influence mRNA processing, turnover and translation rates upon binding of the respective trans-acting factors, such as ELAV1/HuR, PTB or the c-interferon activated inhibitor of translation (GAIT) complex [19–34]. Various microRNAs (miRNAs) have been found to regulate human VEGFA expression post-transcriptionally. MiRNAs are short (,21 nucleotides) noncoding RNAs that generally lead to repression of gene expression upon the binding of recognition motifs that are typically present in the 39-UTR of transcripts. Binding occurs in an antisense fashion which is mainly mediated by a ‘seed’ region which comprises the 59-terminal six to eight nucleotides of the miRNA and exhibits a high degree of complementarity to the target mRNA. Misexpression of miRNAs in hypoxia-induced nasopharyngeal carcinoma-derived CNE cells identified several miRNAs that were able to repress VEGFA expression in vitro, although the corresponding recognition motifs in the mRNAs have not been unambiguously identified [35,36]. One of the two VEGFA internal ribosome entry sites, driving the expression of the VEGF-121 isoform [37], has been shown to be susceptible to regulation by miR-16 [38]. Moreover, miR-16, as well as miR-424, have recently been demonstrated to regulate angiogenic activity in endothelial cells by targeting VEGFA and other angiogenic mediators [39]. Lei et al. identified a feedback loop regulating the adaptation of murine tumor cells to different oxygen concentrations in which hypoxia-inducible factor 1 alpha (HIF-1a) suppresses the expression of miR-20b, which in turn may regulate both HIF-1a and VEGFA expression [40]. Two miRNAs, miR-126 and -205, were shown to regulate VEGFA expression and are implicated in a number of cancers, including lung, prostate and breast cancer [41–46]. Similarly, miR-200c is dysregulated in leiomyomata [47] and endometrial cancers [48], while miR-29b-mediated regulation of VEGFA is implicated in prostate cancers [45]. Jafarifar et al. showed that the binding of the splicing factor hnRNP L to a CA-rich sequence element in the VEGFA and miR-361-5p Expression in Human SCC VEGFA 39-UTR under hypoxia led to competitive displacement of miRNAs miR-297, -299, -567 and -605 and consequently derepression of VEGFA expression in tumor-associated macro- phages [30]. Finally, microRNAs 200b, 93 and 29b were implicated in the non-malignant conditions diabetic retinopathy [49], diabetes [50], and pre-eclampsia [51], respectively. In the present study, we have used miRNA target prediction algorithms and luciferase reporter assays to identify a new microRNA recognition element (MRE) in a downstream con- served region of the VEGFA 39-UTR, and we have confirmed the repressive effect of miR-361-5p on VEGFA expression in vitro with luciferase reporter assays and ELISA. We also found that miR- 361-5p levels were lower in those skin-derived cell lines that express high levels of VEGFA, as well as in SCC tumors compared to healthy skin. These results indicate that miR-361-5p might affect cancer development or progression by modulating VEGFA expression in particular tumor types. Results VEGFA is a Putative Target of microRNA-361-5p The almost 2 kb long sequence of the human VEGFA 39- UTR, the vast majority (.95%) of which is present in all of its known isoforms, contains two regions that are highly conserved among vertebrates, one at its 59- and the other one at its 39-end. While most miRNA recognition elements (MREs) that have been unambiguously shown to be able to affect human or murine VEGFA expression are located in the 59-conserved region [30,38–40,42,50], only miR-126, miR-200b/c, and recently miR-29b have been demonstrated to bind in the ,730 nucleotide downstream conserved region (Figure S1A) [41,43– 45,47–49,51,52]. Both regions are lower in GC content compared to the weakly conserved region separating them (GC% approximately 44, 58 and 28 from 59 to 39). It has also been suggested that the density of functional MREs increases towards both ends of a transcript’s 39-UTR [53]. We therefore wondered whether the 39-conserved region of the VEGFA 39- UTR contains additional MREs that may contribute to VEGFA dysregulation in cancers. In order to find potential candidate miRNAs that may be implicated in the regulation of VEGFA expression, we employed five miRNA target prediction services to search for predicted MREs in the VEGFA 39-UTR: microRNA.org [54], TargetScan [55], DIANA-microT [56], miRDB [57], and MicroCosm [58]. This analysis revealed that only three miRNA/MRE pairs were predicted by all five algorithms (Table S1): Two MREs, predicted to be targeted by miR-29b and miR-205, have already been shown to regulate VEGFA expression [42,45,46,51]; the third MRE, predicted to be regulated by miR-361-5p, is located in the downstream conserved region. RNAhybrid [59] calculated a min- imum free energy of 222.0 kcal/mol for the interaction between miR-361-5p and the MRE located between nucleotides 1604 and 1625 of the VEGFA 39-UTR in transcript NM_001025366 (Figure S1B), which is in the range of other MREs reported for VEGFA [36]. No targets for miR-361-5p have been experimen- tally confirmed so far, but it has been shown that the transfection of a miR-361-5p mimic in hypoxia-induced CNE cells leads to reduced VEGFA protein levels, as determined by ELISA – supporting the idea that this miRNA may regulate VEGFA expression [35,36]. Thus, we chose to focus our studies on this miRNA. MIR361 is encoded on the 6 chromosome, in an intron between exons 9 and 10 of CHM/choroideremia (Rab escort protein 1) and gives rise to two mature miRNA species, miRNA- PLOS ONE | www.plosone.org 2 November 2012 | Volume 7 | Issue 11 | e49568