Proapoptotic effects of miR-204 inhibition in melanoma cell lines and in melanoma models in vivo

MicroRNAs are small molecules of non-coding RNA and they play a relevant regulatory activity in carcinogenesis, during both development and progression steps. In my thesis, I focused on miR-204 which, in melanoma cells, is negatively regulated by BRAFV600E through the ERK pathway. Although it belongs to the same family as miR-211, miR-204 exerts a different biological role. When it is overexpressed, it shows an oncosuppressive function, in particular, it causes a decrease in cell migration/invasion by repressing AP1S2 gene. During my PhD thesis I studied the effects allocated with miR-204 inhibition. Following miR-204 inhibition through the transfection of LNA-204, I unexpectedly observed a significant decrease in cell proliferation, a decrease in cell ability to form colonies and a strong increase in apoptosis in vitro. In addition, I confirmed the antiproliferative and proapoptotic effects in in vivo model systems (zebrafish embryos, nude mice and genetically engineered melanoma mouse model). The biological results are specifically due to miR-204 inhibition (not of miR-211) and they suggest that miR-204 biological role depends on expression levels. Then, I demonstrated that the apoptosis due to miRNA inhibition is dose dependent, using the A375-miR-204-sgRNA melanoma line, in which the amount of endogenous miRNA was reduced by the CRISP-Cas9 technique compared to the control line (A375-AVV1-sgRNA). In addition, I performed the caspase assay and quantified the activity of Caspase 3/7, Caspase-8 and Caspase-9 in order to understand which pathway mediates the apoptotic phenotype. The experimental data suggest that there is not a predominant pathway, but both (intrinsic and extrinsic) are activated at 18h after LNA transfection. I also investigated the molecular mechanism through which the apoptosis occurs. In this regard, at 6h from cell transfection, I measured the increment in p53 protein level in several melanoma cell lines, so I concluded that cell death is related to the p53 signaling. Considering the early induction of p53, I aimed to identify one or more miR-204 specific targets and, for this purpose, I performed an RNA-Seq at 6h post transfection of LNA-204-MM or 204 in A375 and 501 cells. By analysing the sequencing data, I obtained a short list of candidate genes. It includes ATP2A1, IQCN, RCVRN (involved in calcium pathway) and ACTA1, MYH3 (related to adhesion/migration processes). Effectively, at 6h from the transfection of LNA-204, I observed an increment in calcium concentration and a decrease in cell adhesion. To date, I’m proceeding with the characterization of each one in order to understand whether they are direct targets of miRNA or what their role may be in the phenomenon studied. In conclusion, in line with the results obtained and the already known role of genes, I will explore different pathways: the interaction between p53-ATP2A1 at the ER level, the loss of adhesion and the possible involvement of TRPM3 (miR-204 host gene). Furthermore, I will try to understand whether and how calcium signaling affects these different mechanisms.