Numb è un bersaglio diretto del miR-146a

MicroRNAs (miRs) are small (19-21) endogenous noncoding RNAs able to post-transcriptionally downregulate expression of specific target genes by binding to the 3' UTRs of their mRNAs triggering translation repression and/or mRNA degradation. In the last years, miRs have been linked to a variety of physiological and pathological cellular processes. Many studies have focused on cancer, where upregulated miRs can act as oncogenes and downregulated ones can have a tumor suppressor role. Our lab is focusing on the identification and characterization of miRs involved in human malignant melanoma progression. To fulfill our goal, miR expression was evaluated in a human cellular model in which a non-metastatic parental cell line, A375P, was compared with its derived metastatic variants, MA-2 or MC-2, by quantitative Real-Time PCR (qRT-PCR) and microarray analysis. 19 miRs resulted up- or down-modulated in malignant cells. My research project focused on one of the up-regulated small RNAs, miR-146a, previously linked to cancer either as an oncogene or as tumor suppressor. First, I looked for miR-146a putative target genes using: a. the prediction software TargetScan 5.2; b. the Ingenuity Pathway analysis (IPA) platform, the world's largest database for biological networks, able to identify pathways, molecular functions and networks. Second, I crossed the bioinformatics results with the microarray data generated in the lab, obtained from a comparison between miR-146a-overexpressing MA-2 cell line with control cells. By doing so, I identified NUMB as one of the most relevant targets of miR-146a. Therefore, I investigated its role in melanoma progression. Numb is an evolutionary conserved protein that plays critical roles in cell fate determination, endocytosis, cell adhesion and migration, as well as cancer. It is frequently lost in breast cancer, chronic myelogenous laeukemia and salivary gland carcinoma. I investigated if NUMB was a direct target of miR-146a by qRT-PCR, Western Blot (WB) and luciferase reporter assays (NUMB 3'-UTR was cloned downstream of the firefly luciferase coding sequence) and confirmed it. In fact, reduced NUMB mRNA and protein expression, as well as luciferase activity, were found upon miR-146a overexpression in MA-2 cells. Then, I analyzed the biological functions of miR-146a and NUMB by performing in vitro migration, invasion and transendothelial migration assays. I observed that while overexpression of miR-146a impairs all these features, Numb downmodulation (using siRNAs) affects cell migration and invasion, but does not control extravasation. Opposite results were obtained when endogenous miR-146a was down-regulated in MA-2 cells, compared to controls. These evidences suggest that Numb down-regulation, via miR-146a, is relevant in some, although not all, metastatic traits of melanoma cells. Considering that Numb controls Notch pathway and that, in breast cancer, Numb loss causes enhanced Notch signaling and p53 ubiquitination and degradation, leading to malignancy, we hypothesized an involvement of miR-146a in these regulations during melanoma progression. Therefore we analyzed Notch and p53 levels following miR-146a overexpression in MA-2 cells. We found increased Notch, and decreased p53 protein levels, confirming our hypothesis. Our findings suggest an important role for miR-146a and Numb in melanoma progression, and the possible involvement of Notch and p53 signaling open up a new and intriguing line of study.