miR-378a-5p acts as a positive regulator of melanoma progression

Melanoma, the most aggressive form of skin cancer, is frequently associated with alterations in many genes, among which the Bcl-2 oncogene plays an important role in survival, progression, chemosensitivity and angiogenesis. Also microRNAs play an important role in melanoma development and progression affecting tumor proliferation, migration and invasion. HUVEC and a panel of human melanoma cells were used. Western Blot, qRT-PCR, miRNA Microarray, ELISA, vasculogenic mimicry, cell proliferation, clonogenic and invasion assays have been performed. MiR-378a-5p mimic and inhibitor have been used to study the biological and functional role of miR-378a-5p. In silico analysis have been carried out to find putative miRs targets using miRWalk, Diana Tools and Target Scan. To identify putative miRNAs, whose expression could be modulated by Bcl-2, M14 have been transfected with a smart-pool RNA targeting human Bcl-2 mRNA, and a miRNA Microarray has been performed. Four independent experiments indicate that, after Bcl-2 silencing, 13 miRs were significantly downregulated. Among these, miR-378a-5p, has been identified as significantly deregulated in different human melanoma cell lines. Based on the target prediction analysis, we identified and then confirmed, through qRT-PCR, several miR-378a-5p target genes, such as SUFU, STAMBP and KLF9. By performing in vitro experiments, we found that ectopic expression of miR-378a-5p does not significantly affect cell proliferation and clonogenic ability of melanoma cells, while it significantly increases invasion and the expression of MMP2 at protein level. It also facilitates the ability of tumor cells to form de novo vasculogenic structures. We have also evidences that conditioned medium from miR-378a-5p overexpressing melanoma increases the formation of capillary like structures in endothelial cells. To assess the molecular mechanism through which miR-378a-5p induce angiogenesis, ELISA showed that ectopic expression of miR-378a-5p significantly increases VEGF protein secretion, but not IL-8 mRNA expression. Experiments using an antiVEGF neutralizing antibody indicate VEGF implication on miR-378a-5p-induced vasculogenic mimicry. To identify other factors that may be implicated in miR-378a-5p proangiogenic/proinvasive functions, we focused our attention on Sp1/uPAR axis, a pathway we previously demonstrated to be regulated in cancer cells by Bcl-2 under hypoxic conditions. Our experiments indicate that ectopic expression of miR-378a-5p increases uPAR mRNA expression. Surprisingly, this treatment results in a Sp1 reduction, indicating that other transcription factor, other than Sp1, can be responsible for uPAR induction by miR-378a-5p. Based on these evidences, although a deeper understanding of the molecular mechanism involved in Bcl-2 modulation of miR-378a-5p is needed, our findings strongly suggest a proangiogenic and proinvasive role of miR-378a-5p in melanoma cells. Note: This abstract was not presented at the meeting. Citation Format: Maria Grazia Tupone, Marta Di Martile, Simona D9Aguanno, Elisabetta Valentini, Marianna Desideri, Sara Donzelli, Andrea Sacconi, Daniela Trisciuoglio, Giovanni Blandino, Donatella Del Bufalo. miR-378a-5p acts as a positive regulator of melanoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 768.