Circ_0001591 contributes to melanoma cell migration by the sponge of miR-20a-3p and miR-34a-5p, leading to the upregulation of Axl and Fral proteins

Different risk factors are involved in the initiation and progression of melanoma. In particular, genetic and epigenetic pathways are involved in all stages of melanoma and are being exploited in therapeutical approaches. The proteins Axl and Fra1 are overexpressed in melanoma and considered oncoproteins. Circular RNAs and microRNAs are two classes of non-coding RNAs involved in all cancer hallmarks. Circular RNAs are covalently closed single-strand RNA generated by the back splicing of introns, exons, or both. Their main function is to bind microRNAs, thus removing them from their role as inhibitors. MicroRNAs are small non-coding RNAs involved in translation regulation by binding to the 3’UTR of the mRNA target. Circular RNA circ_0001591 has been demonstrated to play a role in melanoma progression and metastasis and presents binding sites for miR-20a-3p and miR-34a-5p, considered tumor-suppressive miRs in melanoma. This thesis investigates the role of circ_0001591 in melanoma migration through the sponge of miR-20a-3p and miR-34a-5p, which increase the expression of Axl and Fra1 oncoproteins. Cell migration was decreased by the inhibition of circ0001591, indicating its possible contribution to melanoma invasion and metastasis by increasing cell mobility. At the same time, miRs expression was increased by circ_0001591 inhibition, demonstrating that circ_0001591 can sponge miR-20a-3p and mir-34a-5p. Luciferase Reporter Assay confirmed the direct interaction between the circRNA and the two miRs. The participation of miR-20a-3p and miR-34a-5p in cell migration was also assessed by wound healing assay, demonstrating a decrease in melanoma migration by the overexpression of the miRs. This effect was reversed by miRs inhibition. By Dual Luciferase Reporter assay, we demonstrate that FOSL1 and AXL genes are a direct target of miR-20a-3p and miR-34a-5p. Following miRs overexpression and inhibition, the expression of mRNAs and proteins was analyzed. The results revealed a decreased expression of AXL mRNA in LM-20 by miR-20a-3p and miR-34a-5p mimic transfections and downregulation of Axl and Fra1 protein expression by both miRs in all melanoma cell lines. This effect was reversed by miRs inhibition. Our results demonstrated the possible contribution of circ_0001591 in melanoma progression, which may be attributed to the sponge miR-20a-3p and miR-34a-5p, which, in turn, cannot downregulate the expression of Axl and Fra1 oncoproteins.