361-3p and the predominant miRNA-361-5p. The locus is highly conserved among placental mammals only, particularly the stem region of the putative precursor miRNA. MicroRNA 361-5p has first been isolated from pancreatic islets by Poy and colleagues [60] and subsequently from neuroblastoma cell lines [61]. In the following, the miRNA has been detected in different tissues and cell types [62–67]. Moreover, in mice the miRNA may be involved in the molecular mechanism underlying insulin resistance and protection thereof [68], and it was found to be differentially expressed in a bleomycin-induced mouse model of lung fibrosis [69]. It is of interest that miR-361-5p is predicted to target mRNAs coding for other proteins acting in angiogenesis (a list of potential miR-361-5p targets compiled from five miRNA target prediction algorithms is given in Table S2; pathways enriched among the Gene Ontology annotations of candidates are shown in Table S3; putative targets acting downstream of VEGFA are highlighted in a pathway map in Figure S2). Potential downstream targets include PKC, Rac, PI3K, NFAT. The pathways most strongly enriched among predicted miR-361-5p targets include upstream regulators of VEGFA expression, such as the EGF and FGF signaling pathways (P = 7.2 6 1026 and 2.6 6 1025, respectively), as well as the VEGF pathway and angiogenesis (P = 0.0085 and 0.0016). MicroRNA-361-5p Targets the VEGFA 39-UTR Due to the generally high degree of interconnectivity within post-transcriptional regulatory networks [70–73], the extensive post-transcriptional regulation that has already been shown to be exerted on the VEGFA transcript [19–52], and the high occurrence of predicted MREs in the conserved region surround- ing the putative MRE (Table S1), it is possible that other trans- binding factors might affect the binding potential of our miRNA of interest. Thus we reasoned that it may be beneficial to preserve potential RNA recognition elements in our experiments. To do this, we cloned the entire downstream conserved region of the VEGFA 39-UTR behind the coding sequence of Renilla luciferase under the control of an SV40 promoter, on a plasmid further encoding a firefly luciferase for normalization purposes (Figure 1A). Additionally, we also generated a mutant of the putative miR-361- 5p MRE, in which three nucleotide residues are deleted (Figure 1B; effective deletion relative to the miRNA seed region = 2 nucleo- tides). In order to avoid competition between the reporter and endogenous VEGFA, we performed the assays in human embryonic kidney (HEK293) cells [74], which express low levels of VEGFA [75]. The ability of HEK293 cells to take up miRNA mimics (see materials and methods) was verified by transfection with increasing amounts of a Cy3-labeled control oligonucleotides, followed by flow cytometry analysis (Figure S3), revealing that.90% of the cells take up the labeled mimics across the whole range of concentrations in a dose-respondent fashion. When overexpressing the miR-361-5p mimic together with the wild type VEGFA 39- UTR luciferase reporter, relative Renilla activity was decreased by approximately 25% compared to the control precursor (P = 0.0327; unpaired t-test, two-tailed), as well as the mutated reporter (P = 0.0263; unpaired t-test, two-tailed), which did not change relative to the control (Figure 1C). Although the co- transfection of wild type and mutant reporters with miR-361-5p antisense inhibitor or control did not lead to significant differential luciferase activities, these data indicate that the putative miR-361- 5p MRE possesses regulatory potential, and that it is subject to regulation by miR-361-5p. VEGFA and miR-361-5p Expression in Human SCC Endogenous VEGFA Expression is Regulated by microRNA-361-5p To check whether endogenous VEGFA levels could be affected by miR-361-5p, we chose to alter levels of the miRNA in epidermoid squamous cell carcinoma-derived A431 cells [76] and keratinocytes derived from normal skin that transformed sponta- neously in vitro (HaCaT) [77] – both of which are known to express high levels of VEGFA, as well as low levels of miR-361-5p (see the following paragraph). First, the efficiencies of transfecting these cells with miRNA mimics and antisense inhibitors were tested with increasing concentrations of Cy3-labeled control oligonucleotides and monitored by flow cytometry (Figure S3). Oligonucleotides were incorporated by more than 90% of the cells in all conditions, with fluorescence intensities increasing in a dose-respondent manner. We then determined VEGFA levels in the culture supernatants of both cell lines transfected with different amounts of miRNA-361-5p mimic or control using ELISA (Figure 2A/B). Baseline levels of secreted VEGFA of mock-transfected cells were approximately 31766585 and 13146152 pg/mL per 24 hours, for A431 and HaCaT cells respectively. VEGFA levels were significantly decreased in A431 cells (up to ,30% when transfecting 10 nM; P = 0.0063; unpaired t-test, two-tailed; Figure 2A), while we only observed a slight decrease in VEGFA levels in HaCaT cells (up to ,11% when transfecting 30 nM; P = 0.0502; unpaired t-test, two-tailed; Figure 2B) when comparing transfection of miR-361-5p mimic and control. Conversely, VEGFA levels were not affected in A431 cells transfected with increasing amounts of miRNA-361-5p antisense inhibitor when compared to cells transfected with a control (Figure 2A), while in HaCaT cells we detected elevated VEGFA levels for all tested antisense inhibitor concentrations (up to ,39% when transfecting 10 nM; P = 0.0150; unpaired t-test, two-tailed; Figure 2B). Next we tested the effects of miR-361-5p mimics and antisense inhibitors in a setting where VEGFA expression is induced by treatment with TNF-a (10 ng/mL, 24 h; Figure 2C/D) [16]. As expected, baseline levels of VEGFA after 24 hours were increased by 18% (A431, 37456266 pg/mL) and 138% (HaCaT, 31236254 pg/mL) when compared to uninduced states (compare above). Importantly, significant changes in secreted VEGFA levels were found for miR-361-5p mimics and antisense inhibitors in A431 (,26/24% decrease/increase for mimics and antisense inhibitors, respectively; P = 0.0045 and 0.0181; unpaired t-test, two-tailed; Figure 2C) and HaCaT cells (,19/17% decrease/ increase for mimics and antisense inhibitors, respectively; P = 0.0150 and 0.0233; unpaired t-test, two-tailed; Figure 2D), when compared to the corresponding control oligonucleotides. Taken together, these findings demonstrate that miR-361-5p affects the levels of secreted VEGFA, further suggesting that the miRNA is able to repress endogenous VEGFA expression in vitro. VEGFA and miR-361-5p Expression Levels are Inversely Correlated in Skin-derived Cells in vitro Next, we used quantitative reverse transcription PCR (qRT- PCR) to determine the expression levels of endogenous miR-361- 5p and VEGFA mRNA in four skin-derived human cell lines: (1) the VEGFA-secreting epithelial cell lines A431 and HaCaT; (2) primary dermal lymphatic (LEC) and blood vascular (BEC) endothelial cells, which are targeted by VEGFA but express only low levels of it themselves [78,79]. While miRNA expression did not differ significantly within the two groups of cells (fold difference between A431 and HaCaT = 1.04+0.97–0.50; fold difference between BEC and LEC = 1.94+3.77–1.28), miR-361- 5p levels were significantly higher in the endothelial cells PLOS ONE | www.plosone.org 3 November 2012 | Volume 7 | Issue 11 | e49568