render the cells unresponsive to miR-361-5p inhibition. Likewise, the absence of an effect of miR-361-5p inhibition on luciferase reporter activity in HEK293 cells could also be explained by a relatively low MRE occupancy. However, data obtained from stimulated A431 cells, which express slightly more VEGFA than HaCaT cells, yet proved responsive to miR-361-5p inhibition, speaks against such a simple model. Moreover, in this scenario the impact of increased miR-361-5p levels in unstimulated A431 and miR-361-5p inhibition in unstimulated HaCaT cells should be proportional to the amount of added miRNA mimic or antisense inhibitor. The absence of such a miRNA dose-response could indicate that one or more additional factors differentially modulate miR-361-5p activity in the two cell lines, systemically or specifically, by influencing the availability, accessibility or func- tionality of the miRNA or its recognition element. The large set of known post-transcriptional regulators of VEGFA expression [19– 52], as well as the extremely high density of predicted MREs in the downstream conserved region of the VEGFA 39-UTR (0.41 MREs/nucleotide, compared to 0.29 and 0.14 for the upstream conserved and non-conserved regions, respectively, Table S1), add particular weight to such a hypothesis, as it implies the potential for a high degree of competition or similar cooperative effects between miRNAs, or between miRNAs and RNA-binding proteins. Indeed, such cooperation has already been observed for other miRNAs regulating VEGF expression in vitro [35]. Various studies have also demonstrated functional interplay between miRNAs and RNA-binding proteins on a number of targets [72], including VEGFA [30], and it has been proposed that such crosstalk may be widespread [83]. Furthermore, the localization of the MRE so close to the mRNA’s 39-terminus may render it particularly prone to in- accessibility due to degradation or alternative polyadenylation. Regarding the latter, it has recently been proposed that pro- liferating cells may employ alternative polyadenylation to shorten the 39-UTRs of transcripts and thus escape miRNA- and RNA- binding protein-mediated post-transcriptional repression [84]. Indeed, it was found that the VEGFA transcript uses two different polyadenylation sites in mice, although no differential usage of the signals was observed between normoxic and hypoxic conditions [85]. Similarly, our data indicate an apparent difference in expression levels between the coding region and the downstream region of the 39-UTR within but not in between both clinical sample groups, suggesting that alternative polyadenylation may indeed limit the availability of the miR-361-5p MRE (Figure S4). However, slight differences in the amplification efficiencies of the used gene expression assays may also account for these observa- tions. Further studies into the differential regulation of VEGFA 39- UTR length, the interplay between miRNAs, as well as the crosstalk with other post-transcriptional regulators may shed light on these issues. It cannot be ruled out that the ELISA results are due to secondary effects on non-targets or other miR-361-5p targets acting in the same pathway, which could in turn lead to altered VEGFA production/secretion. Indeed, while the reliability of miRNA target prediction software is often questionable at the level of individual targets, the strong enrichment of VEGFA-related pathways among predicted miR-361-5p targets (Table S2, Table S3, Figure S2) indicates that the miRNA may have potential roles in regulating angiogenesis and other VEGFA-related functions, both upstream and downstream of the VEGFA/VEGF receptor axis. We have shown that miR-361-5p is expressed in a number of cell types derived from human skin. Interestingly, the epithelial, VEGFA-secreting cells (A431 and HaCaT) express significantly VEGFA and miR-361-5p Expression in Human SCC lower levels of this miRNA than the endothelial cells ‘‘targeted’’ by VEGFA (LEC and BEC). This may indicate a potential role for miR-361-5p in upholding the physiological balance of VEGFA levels in the skin. Furthermore, we have shown that levels of miR- 361-5p, but not those of the known VEGFA-regulating miRNAs miR-20b, -34a, -93, -126, and -205, inversely correlate with VEGFA expression in SCC compared to healthy skin samples, corresponding to previously reported findings [86]. Therefore, out of the assayed microRNAs, miR-361-5p seems most likely to contribute to elevated VEGFA mRNA levels in this type of cancer. It is well established that VEGFA, secreted by cultured keratinocytes, potently induces the proliferation of endothelial cells [87], implying that suppressors of VEGFA secretion may interfere with VEGFA-related functions in target cells. Moreover, previous findings in an experimental SCC model, demonstrating that elevated VEGFA levels were associated with increased tumor cell invasion and angiogenesis [10], indicate an indirect mecha- nism by which miR-361-5p might promote SCC progression. To gain first insights into possible functions of miR-361-5p, we analyzed the impact of altered miR-361-5p levels on proliferation (Figure S5A/B) and migration (Figure S5C/D) of A431 and HaCaT cells. However, the observed slight effects are likely not correlated with its targeting of VEGFA and may be the result of targeting other cancer related genes. Nevertheless, the presented data may provide a starting point for future studies investigating the potential roles of miR-361-5p on migration, proliferation and angiogenesis in relevant cell lines and SCC models. Next to a characterization of the miRNA’s functions in vitro, it may also be of interest – given the importance of VEGFA-related processes for wound healing and tissue regeneration – to study its function in an appropriate in vivo model (e.g. cutaneous wound healing). With regard to the potential involvement of microRNA 361-5p in the etiology of SCC, an important question raised by our study is how the expression of the miRNA is regulated. One possibility is certainly that miR-361-5p transcription is dependent on its ‘‘host gene’’ CHM. However, while the levels of miRNA and ‘host gene’ expression correlated fairly well among the clinical samples, CHM and VEGFA levels did not. It is possible that additional regulation at the various steps of miRNA processing, as has been shown for a wide range of miRNAs [80], could lead to the observed discrepancies in the correlation of the assumed primary transcript (CHM) and the mature miRNA. Another possibility is that the miRNA bears its own promoter elements that could uncouple miRNA transcription from that of CHM. However, we are not aware of any literature on the transcriptional regulation of CHM. Therefore, detailed studies into the regulation of miRNA 361 should consider analyzing primary transcripts levels as well as those of miR-361-5p and -3p, their precursors, as well as CHM/ Rab escort protein 1 protein levels. To our knowledge, CHM has not been implicated in either SCC or other skin-related diseases. However, there is some indication about a possible involvement of VEGFA in the etiology of choroideremia, a degenerative disease of the retina associated with the full or partial deletion of CHM and predominantly occurring in males: Vascular abnormalities in the endothelial cells of choroideremia patients have been previously reported [88]. A more recent study further identified VEGFA as one of a group of factors that is frequently expressed in a variable manner in the monocytes of choroideremia patients, and its levels correlated with disease severity [89]. Interestingly, many of the observed mutations/deletions in the CHM alleles of patients also affect miR-361-5p [90]. Loss-of-function mutations of miR-361-5p may thus potentially contribute to the observed variations in VEGFA levels. Finally, miRNA 361 is located on the 6 chromosome, which encodes 10% of all human miRNAs [91]. PLOS ONE | www.plosone.org 8 November 2012 | Volume 7 | Issue 11 | e49568