VEGFA and miR-361-5p Expression in Human SCC Figure 1. Mutation of the putative recognition element abolishes miRNA-361-5p-mediated regulation of a VEGFA 39-UTR reporter. (A) Schematic representation of the luciferase reporter constructs, indicating the VEGFA 39-UTR fragment fused to Renilla luciferase, the predicted miRNA recognition element (MRE) for miR-361-5p, and the firefly luciferase gene used for normalization. (B) Sequence alignment of miR-361-5p with the wild type (wt) VEGFA 39-UTR and mutated reporter. The seed sequence (bold) and mutated residues (red) are highlighted. (C) HEK293 cells were co-transfected with either of the indicated luciferase reporters and a miR-361-5p mimic, antisense inhibitor, or the respective control. Data were obtained from three independent experiments, performed in triplicate. For each experiment, mean ratios of Renilla and firefly luciferase activities were normalized to those of psiCHECK-2-transfected cells, then to Pre- or Anti-miR control-transfected cells. Mean values of three independent experiments 6 S.D. are plotted. Two-tailed, unpaired t-tests were used to calculate P values (single asterisks denote P values,0.05). doi:10.1371/journal.pone.0049568.g001 compared to the VEGFA-expressing epithelial cells (fold differ- ences between LEC/BEC and A431/HaCaT ranging from 23.72 to 47.82; P values between 0.0064 and 0.0120; unpaired t-test, two tailed; Figure 3A). Conversely, while VEGFA mRNA levels differed only slightly within the two groups of cells (fold difference between A431 and HaCaT = 3.86+1.48–1.07; P = 4.061028; fold difference between LEC and BEC = 1.46+5.12–1.13), the epithe- lial cells contained considerably higher amounts of VEGFA mRNA compared to the endothelial cells (fold differences between A431/HaCaT and LEC/BEC ranging from 70.43 to 378.41; P values between 0.0019 and 0.0101; unpaired t-test, two tailed; Figure 3B). With a Spearman’s rank correlation coefficient of the DCT values of r = 20.80 (two-tailed) across all cell lines, the expression levels of miR-361-5p and VEGFA correlated well in an inverse manner, although this correlation did not reach signifi- cance. In summary, these data indicate that miR-361-5p is expressed in all of the tested skin-derived cell lines, with the highest levels occurring in endothelial cells. Considering the observed relation between miRNA and VEGFA levels, it is possible that miR-361-5p might play a role in maintaining the functional balance of differential VEGFA expression between epithelial and endothelial cells in the skin. PLOS ONE | www.plosone.org 4 November 2012 | Volume 7 | Issue 11 | e49568