• ncRNAs
• circRNAs
• melanoma

Circular RNAs (circRNAs) belong to the class of noncoding RNAs (ncRNAs), but they are relatively little studied compared to other ncRNAs. At first considered the result of splicing errors, it has now been shown that circRNAs are evolutionarily conserved, and they have roles in cellular functions. CircRNAs lack 3’ and 5’ ends and, since they are covalently closed molecules, are highly stable and resistant to degradation by RNA exonucleases. Their primary function was proposed to be as miRNAs sponges, but it has been shown that circRNAs can interact with proteins to affect their function and localization, modulate the expression of their parental gene, and some can even be translated into proteins. Roles for circRNAs have been shown in multiple diseases including cancer, and they are being studied as potential cancer drivers and biomarkers. We hypothesized that circRNAs can play pro-tumorigenic roles also in melanoma progression. To investigate this, we performed RNA sequencing and found numerous circRNAs exhibiting differential expression between human melanocytes and melanoma cell lines. Among those, we selected circRNAs with the greatest fold-change in expression for validation in additional cell lines using qRT-PCR. The qRT-PCR for detecting circRNAs requires the use of divergent primers spanning the back-splice junction, the region of the circRNA where the two ends of the back-spliced linear transcript converge leading to the circular form. This validation has led to the selection of circPAX3, which is found to be downregulated in melanoma cell lines. We hypothesize that circPAX3 may have anti-tumorigenic functions and to test this hypothesis we tested the effect of modulating circPAX3 expression on melanoma cell behavior in migration, invasion, proliferation, focus formation and soft agar assays.

The two parallel experiments to test if circPAX3 differential expression leads to different phenotypes are its overexpression and its silencing. The overexpression of circPAX3 in melanoma cell lines is achieved by transfecting cells with a plasmid carrying a transposon that contains the circPAX3 sequence between two ZKSCAN introns, known to trigger the process of back-splicing. The transposon, mediated by the concurrent expression of the PiggyBac transposase using another plasmid, integrates into the genome and its transcription allows the production of the circRNA and the blasticidin deaminase resistance gene. Transfected cells are selected using blasticidin, so that just the cells that integrated the transposon in the genome can survive. These cells are then used for performing cells assays. On the other hand, the silencing of circPAX3 is achieved by using siRNAs designed over the back-splice junction, so that they can target the circRNA but not its linear counterpart. After siRNAs transfection, cell assays were performed to assess the silencing.

SkMel28 melanoma cells silenced for circPAX3 show a more aggressive phenotype, especially in migration and proliferation, while the overexpression of circPAX3 in WM164, WM793 and A375 melanoma cell lines reduces the aggressiveness of the cells, slowing migration and invasion compared to the negative control cells expressing spGFP. Moreover, the circPAX3-overexpressing WM164 and A375 cells show a significant reduction of their proliferation rates, with a more contact-dependent behavior as they are less capable of sustained growth in low-cell density assays and in lack of substrate contact, as the growth in suspension immersed in agar.

All these results are promising, and further functional characterization of the mechanism of action of circPAX3 is needed.