VEGFA and miR-361-5p Expression in Human SCC Figure 3. VEGFA and miR-361-5p expression levels are inversely correlated in human skin-derived cell lines. qRT-PCR analysis of miR- 361-5p (A) and VEGFA (B) expression in A431, HaCaT, primary human dermal lymphatic (LEC) and blood endothelial cells (BEC). Fold differences 6 S.D. in expression levels with regards to the references (RNU6B and ACTB, for miRNA-361-5p and miRNA-361-5p, respectively) are plotted. Data represent at least three independent experiments performed in duplicate. Two-tailed, unpaired t-tests were used to calculate P values (one, two and three asterisks denote P values,0.05,,0.01, and,0.001, respectively). doi:10.1371/journal.pone.0049568.g003 MicroRNA-361-5p Levels are Decreased in Cutaneous Squamous Cell Carcinoma Having established that miR-361-5p levels are lower in those skin-derived cell lines that exhibited higher levels of VEGFA expression in vitro, we hypothesized that its expression may be lower in SCC, and that it may thus contribute to the initiation or maintenance of high VEGFA expression, either directly or indirectly. We therefore measured the expression of miR-361-5p and several other VEGFA-regulating miRNAs in five samples of SCC obtained from patients and in five healthy skin samples using qRT-PCR. First we investigated whether VEGFA expression was indeed increased in the SCC samples by assessing VEGFA mRNA levels with two different assays, one for exon 3 and the other one for the downstream conserved region in the 39-UTR. Interestingly, we found that the VEGFA 39-terminus was expressed at significantly lower levels than the coding region (fold difference between VEGFA exon 3 and VEGFA 39-terminus assays of 2.72+1.34– 0.90; P = 0.0098; unpaired t-test with Welch’s correction, two- tailed; P = 0.0115; Mann-Whitney U = 17, n1 = n2 = 10, two- tailed; Figure S4). As expected, we found that VEGFA mRNA levels were around two-fold higher in the SCC samples compared to healthy control samples (fold difference between SCC and healthy skin for the exon 3 assay: 2.27+2.61–1.22; P = 20.0472; unpaired t-test with Welch’s correction, two-tailed; P = 0.0556; Mann-Whitney U = 3, n1 = n2 = 5, two-tailed; fold difference between SCC and healthy skin for the 39-terminal assay: 2.12+4.50–1.44; P = 0.1846; unpaired t-test with Welch’s correc- tion, two-tailed; P = 0.2222; Mann-Whitney U = 6, n1 = n2 = 5, two-tailed; Figure 4A and S4B). Moreover, data correlated very well for both assays (r = 0.83, P = 0.0015; Spearman’s rank correlation, one-tailed; Figure 4B). Next, we measured the expression of miR-361-5p and the known VEGFA-regulating miRNAs miR-20b, miR-34a, miR-93, miR-126 and miR-205. In order to gain first insights into the regulation of miR-361-5p, we also determined the expression levels of the miRNA’s ‘host gene’ CHM. In normal skin samples, the average expression levels of miR-20b (,62-fold down) and miR-205 (,51-fold up) strongly deviated from that of the reference RNA (RNU6B), while for all other miRNAs the difference stayed within an order of magnitude (Figure S4A). Of note, miR-361-5p levels (,3.6-fold lower than RNU6B) were very consistent between samples (Figure S4A) and correlated fairly well with CHM mRNA levels (r = 0.53, P = 0.0587; Spearman’s rank correlation, one-tailed; data not shown). In the SCC samples, miR-361-5p and CHM levels were significantly decreased compared to healthy skin samples (fold difference between SCC and healthy skin for the miR-361-5p assay: 0.44+0.33–0.19; P = 0.0220; unpaired t-test with Welch’s correction, two-tailed; P = 0.0159; Mann-Whitney U = 1, n1 = n2 = 5, two-tailed; fold difference between SCC and healthy skin for the CHM assay: 0.40+0.53–0.23; P = 0.0456; unpaired t-test with Welch’s correc- tion, two-tailed; P = 0.0952; Mann-Whitney U = 4, n1 = n2 = 5, two-tailed), whereas all other tested miRNAs either did not exhibit considerably reduced expression levels or, as seen for miR-126, even appeared to be increasingly expressed (fold difference between SCC and healthy skin of 3.72+10.99–2.78; P = 0.0699; unpaired t-test with Welch’s correction, two-tailed; P = 0.0952; Mann-Whitney U = 4, n1 = n2 = 5, two-tailed; Figure 4A and S4B). Importantly, miR-361-5p levels exhibited the strongest inverse correlation with VEGFA levels across all samples (r = -0.58 and -0.60, P = 0.0408 and 0.0333 for the exon 3 and 39-terminal assays, respectively; Spearman’s rank correlation, one-tailed; Figure 4B). No other miRNA passed an r #20.5 threshold. Notably, CHM did not correlate with VEGFA expression (r = 0.05 and 0.09, P = 0.4405 and 0.4014; Spearman’s rank correlation, one-tailed; for the exon 3 and 39-terminal assays, respectively). In summary, we found that out of a panel of six miRNAs targeting VEGFA, only miR-361-5p levels were decreased in SCC expressing high levels of VEGFA, indicating that miR-361-5p dysregulation could contribute to the observed elevated VEGFA levels in SCC. Although we did not observe a similar inverse correlation between CHM and VEGFA mRNA levels, the correlation between CHM and miR-361-5p levels may be an indication that transcription of the miRNA precursor is dependent PLOS ONE | www.plosone.org 6 November 2012 | Volume 7 | Issue 11 | e49568