Identification of functional miRNA interactions in Malignant Melanoma progression

According to the World Health Organization (WHO), Malignant Melanoma is the most aggressive form of skin cancer. Melanoma accounts for only about 4% of skin cancer cases but for as many as 74% of all deaths caused by skin cancers. The development of regional and/or distant metastases and the lack of promising therapies for the treatment of Melanoma are the principal causes of extremely poor survival of Melanoma patients. Recently, enormous efforts are taken to unravel the molecular mechanisms that lead to tumour development and metastasis. A new class of small non-coding RNAs, the microRNAs (miRNAs), has been involved in tumour progression and metastasis. MiRNAs are able to modulate the expression of specific target genes binding to their 3’-UTRs through Ago proteins complex and usually causing transcript degradation. The ability of miRNAs to achieve simultaneous fine-tuning of numerous different targets makes them a fundamental system for cell regulation but, despite their biological importance, the identification of their targets is still a challenging research task. The aim of my project is the identification of real functional miRNA-target interactions involved in pathways that control Melanoma metastasis. The experiments were performed using the metastasis model developed by Xu and colleagues (Xu et al., 2008) composed by a low metastatic Melanoma cell line (LMCs), its derivative high metastatic cell lines (HMCs) and the lung metastases (Mets) arisen after these cell lines are injected into immunodeficient mice. This model mimics the behaviour of cancer cells during metastasis progression. To reach proposed aims, I have set up a meta-analysis approach first and a genome wide biochemical approach later. The combination of the two approaches have been allowed the experimental validation of results obtained by meta-analysis and the identification of new miRNA interactors. I first performed miRNA microarray experiments on LMCs, HMCs and Mets samples observing that miRNA expression profiles are able to distinguish the three different grade of metastasis. Since the evidence of miRNAs involvement in metastasi, I used a meta-analysis approach to evidence the influence of miRNA in pathways involved in the extravasation processes: WNT signalling pathway, Adherens Junction pathway, and VEGF signalling pathway. Moreover, I identified important miRNA clusters (e.g. miR-106a~363 cluster) involved in the regulation of metastatic hallmarks such as the cell protrusion formation, cell-cell signaling, and the tissue vascularization. In order to validate all the interactions identified by meta-analysis, I set up conditions for the simultaneous experimental identification of miRNA-target interactions through the high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HiTS-CLiP). This method uses ultraviolet irradiation to covalently crosslink RNA–protein complexes, in our case Ago proteins, that are in direct contact (approximately over single ängstrom distances) within cells. Through the immunoprecipitation complexes are purified allowing the identification of interacting RNA through high-throughput sequencing methods. This technique validated several miRNA-mRNA interactions identified by meta-analysis and by literature. In particular, has been observed the involvement of miR-106a in Adherens junction and VEGF signaling pathways where seems to act as metastasis suppressor inhibiting pro-metastatic genes as WAVE3 and VEGFA. Moreover, sequencing analyses have confirmed the metastatic powerful for miR-214 and let-7c in Melanoma metastasis. The importance of miR-214 was confirmed by results obtained by our collaborator Dr. Taverna of University of Torino, while I used let-7c inhibition to confirm that it interacts with two targets (FNDC3B and FOXN) identified by AGO-Hits-CLIP experiment and that can be studied in future functional studies. Finally, I have also discussed the involvement of a new class of miRNA regulators: the long non-coding RNAs (lncRNAs). I identified a lot of lncRNAs with the ability to regulate miRNA availability. Taken together, these findings demonstrate the strong involvement of miRNAs in metastasis. In fact, several functional miRNA-transcript interactions could regulate extravasation pathways during the Melanoma cells spreading. My experimental approach successfully guides us towards important biological results with interesting therapeutic implications in Melanoma