The expression levels of microRNA-361-5p and its target VEGFA are inversely correlated in human cutaneous squamous cell carcinoma

Abstract

Vascular endothelial growth factor A (VEGFA) plays a key role in the angiogenesis of human skin. Elevated levels of VEGFA are associated with several pathological conditions, including chronic inflammatory skin diseases and several types of skin cancer. In particular, squamous cell carcinoma (SCC) of the skin, the second most common skin cancer in the general population, is characterized by invasive growth, pronounced angiogenesis and elevated levels of VEGFA. The processing, turnover and production of VEGFA are extensively regulated at the post-transcriptional level, both by RNA-binding proteins and microRNAs (miRNAs). In the present study, we identified a new miRNA recognition element in a downstream conserved region of the VEGFA 3'-UTR. We confirmed the repressive effect of miR-361-5p on this element in vitro, identifying the first target for this miRNA. Importantly, we found that miR-361-5p levels are inversely correlated with VEGFA expression in SCC and in healthy skin, indicating that miR-361-5p could play a role in cancers.

Figure 1. Mutation of the putative recognition element abolishes miRNA-361-5p-mediated regulation of a VEGFA 3′-UTR reporter.

(A) Schematic representation of the luciferase reporter constructs, indicating the VEGFA 3′-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 3′-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 ± S.D. are plotted. Two-tailed, unpaired t-tests were used to calculate P values (single asterisks denote P values<0.05).

Figure 2. Impact of altered miRNA-361-5p levels on secretion of endogenous VEGFA.

A431 and HaCaT cells were transfected with the indicated concentrations of miR-361-5p mimics, antisense inhibitors, or controls, either in the presence (A, B) or absence (C, D) of TNF-α. Cellular supernatants were analyzed for VEGFA protein content using a human VEGFA ELISA. Three independent experiments were performed in triplicate. Mean values ± S.D. from a representative experiment are plotted. Two-tailed, unpaired t-tests were used to calculate P values (one and two asterisks denote P values<0.05 and<0.01, respectively).

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 ± 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).

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 C_T 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 ± 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.