Melanoma is a high-grade, poorly differentiated malignant tumor of pigment-producing cells, accounting for more than 75% of the skin cancer related deaths. MicroRNAs (miRNAs) are short non-coding RNA molecules that affect various cancer related functions with clinical importance and significance.
MiR-20a, which is a member of the miR-17/92 cluster family, was up-regulated in a highly aggressive cell line as compared to an isogenic poorly aggressive cell line and was therefore selected as a possible oncogenic miRNA. Paradoxically, over-expression of miR-20a in melanoma cellsinhibitedaggressive cancer featuresin vitro, including net proliferation and invasion.
A comparative Affymetrix whole human genome microarray was performed on mock- and miR-20a- transduced melanoma cells. Notably, 623 genes were down-regulated in the miR-20a over-expressing cells. Crossing of the list of down-regulated genes (microarray data) with the list of predicted targets of miR-20a pointed on 56 genes as plausible miR-20a targets. By 3’UTR cloning into luciferase vector, introducing abrogating mutations into the putative miR-20a binding sites, and using luciferase assays, we positively identified one new direct target for miR-20a, ETV1. This gene was previously reported to facilitate tumor-promoting features such as migration and proliferation.
We will study its role in melanoma using the following outline: a) test the expression of ETV1 at the mRNA and protein levels following overexpression of miR-20a using qPCR and Western blot; b) pheno-copy by knocking down ETV1 with specific shRNA in melanoma cells, followed by specific rescue using shRNA-resistant ETV1 construct. The cells will be tested in proliferation, cell cycle, invasion andin vivotumorigenicity; c) miR-20a shares an identical seed region with miR-17, which exerts tumor-facilitating properties. We will study the differential regulation of ETV1 by miR-20a and miR-17; d) study the expression of ETV1 in primary cultures of metastatic melanoma cells and in melanoma progression tumor microarrays.
