Differential regulation of aggressive features in melanoma cells by members of the miR-17-92 complex

The various roles of microRNAs (miRNAs) in controlling the phenotype of cancer cells are the focus of contemporary research efforts. We have recently shown that miR-17 directly targets the ADAR1 gene and thereby enhances melanoma cell aggressiveness. miR-17 and miR-20a belong to the miR-17/92 complex, and their mature forms are identical except for two non-seed nucleotides. Nevertheless, here we show that these two miRNAs carry markedly different effects on melanoma cells. A strong positive correlation was observed between the expression of miR-17 and miR-20a among various melanoma cultures. Luciferase assays showed that miR-17 but not miR-20a directly targets the 3' untranslated region of the ADAR1 gene. Ectopic expression of these miRNAs in melanoma cells differentially alters the expression of five exemplar TargetScan-predicted target genes ADAR1, ITGB8, TGFBR2, MMP2 and VEGF-A. Whole genome expression microarrays confirm a markedly differential effect on the transcriptome. Functionally, over-expression of miR-20a but not of miR-17 in melanoma cells inhibits net proliferation in-vitro. The differential functional effect was observed following ectopic expression of the mature miRNA or of the pre-miRNA sequences. This suggests that the two non-seed nucleotides dictate target sequence recognition and overall functional relevance. These miRNAs are clearly not redundant in melanoma cell biology. miR-20a or miR-17 were cloned into pQCXIP and transduced into PAG (C81-61) cells. The cells were subjected to RNA extraction and hybridization on Affymetrix whole genome expression microarrays and compared with control cells (mock transduction).