VEGFA and miR-361-5p Expression in Human SCC Figure 4. Relative changes in expression of selected mRNAs and mature miRNAs in healthy skin and SCCs. For each group, five samples were analyzed by qRT-PCR, representing ten individuals. Experiments were performed in quadruplicates. Data are based on CT values normalized to ACTB and RNU6B for mRNAs and miRNAs, respectively. (A) Fold differences in expression relative to average of expression in healthy skin samples 6 S.D. are indicated for each assay on a logarithmic scale. The Mann-Whitney U test was used to calculate P values (two-tailed; the single asterisk denotes a P value,0.05). (B) Based on the normalized expression levels, Spearman rank correlation coefficients (r) between relative expression levels of VEGFA and each of the indicated miRNAs were calculated over all samples. P values (one-tailed) are indicated. doi:10.1371/journal.pone.0049568.g004 on CHM transcription. Nevertheless, additional regulation at the miRNA processing step may occur and contribute to the non- correlated response of CHM and VEGFA transcripts [80]. Discussion In this study, we have identified microRNA 361-5p as a novel potential regulator of VEGFA expression. Using luciferase re- porter assays, we have shown that miR-361-5p can target the VEGFA message via a recognition motif that is located in the downstream conserved region of the VEGFA 39-UTR. Of note, an additional potential miR-361-5p MRE (located between nucleotides 1900 and 1921 of the 39-UTR) was predicted by microRNA.org, and could contribute to the miRNA’s observed repressive potential, but was not analyzed by mutational analysis as part of this study because of the low prediction coverage and weaker calculated free minimum energy (-12.8 kcal/mol; RNAhy- brid). Consistent with findings indicating that the regulatory impact of altered miRNA levels on endogenous protein levels of targets is often weak [81,82], we have also observed mild inhibitory effects of miRNA 361-5p on the levels of secreted VEGFA. A difference in VEGFA miR-361-5p MRE occupancy by endogenous miR-361-5p between HaCaT and A431 cells could potentially explain the differential effects of miR-361-5p mimic or antisense inhibitor on the levels of secreted VEGFA in unstimulated cells: A high occupancy (‘saturation’) of the MRE in HaCaT cells, which express similar miR-361-5p but lower VEGFA levels compared to A431, could account for the absence of a significant decrease in VEGFA secretion upon the addition of miR-361-5p mimic. Consistently, stimulated HaCaT cells, which secreted.2-fold higher levels of VEGFA compared to unstimu- lated cells, responded to elevated levels of miR-361-5p. Converse- ly, a low occupancy of the MRE in unstimulated A431 cells could PLOS ONE | www.plosone.org 7 November 2012 | Volume 7 | Issue 11 | e49568