miR-146a nella progressione del melanoma

MicroRNAs (miRNAs) are endogenous small non-coding RNAs which function at the post-transcriptional level as negative regulators of gene expression by binding at the 3' UTR of mRNA targets and preventing protein translation or promoting mRNA degradation. More than 700 miRNA genes have already been identified in the human genome, and since miRNAs coordinate the expression of multiple genes, they are responsible for regulation of the most important cellular processes, like growth, proliferation, development, differentiation and apoptosis. The abnormal expression or alteration of miRNAs has been shown to contribute to several human malignancies, including cancer where miRNAs can act as oncogenes or tumor suppressors. In the laboratory, several miRNAs involved in human malignant melanoma have been identified. I focused on miR-146a, which we found to be upregulated in the MA2 metastatic melanoma cells compared to the A375P non metastatic parental counterparts or in other highly aggressive melanoma cell lines, such as Mel1300 cells. From literature, miR-146a seems to have a role either as oncogene or as tumor suppressor (Lin, Chiang et al. 2008, Xu, Zhu et al. 2008) depending on the tumor considered and consequently on its targets. In order to understand the effect of miR-146a in melanoma, cells were transduced (for pre-miR-146a or for miR-146a-sponges) or transfected (pre- or anti-miR-146a oligos) with the appropriate expression vectors or oligos to obtain overexpression or downmodulation for miR-146a. Here, we investigated its biological functions by analyzing cell proliferation, anchorage-independent growth and cell migration. We found that miR-146a overexpression increased cell proliferation and colony formation in soft agar, but decreased cell migration. Conversely its downmodulation led to the opposite effects. In order to identify miR-146a putative targets we used the TargetScan 6.2 and Diana Tools TarBase algorithm, we referred to the literature and we analyzed their functions using the Ingenuity Pathway Analysis (IPA) system. We focused on two targets, NRAS and ROCK1. Both of them are known to regulate formation of cellular protrusions, cell proliferation and cell movement or invasion and are involved in melanoma progression. ROCK1 is an effector of the RhoA family. It controls actomyosin contractility leading to cytoskeletal stress fiber formation, focal adhesion complex formation, smooth muscle contraction, and cell migration. NRAS is a membrane-bound protein with GTPase activity that functions as regulatory element in the signal transduction of numerous hormones, cytokines, and growth factors, affecting cell proliferation, differentiation, migration, and apoptosis. By qRT-PCR, WB analysis and luciferase assays, we demonstrated that overexpression of miR-146a correlates with the downmodulation of these two targets, instead miR-146a downregulation leads to increased levels. Our findings suggest a role for miR-146a in melanoma progression. In particular, this small RNA induces tumor cell growth but prevents metastasic traits. In the future we would like to understand if the downmodulation of ROCK1 and NRAS by miR-146a is responsible for the arrest of cell migration and metastasis formation.