Caratterizzazione dell'RNA circolare CircFARSA in linee cellulari di Tumore al Polmone Non a Piccole Cellule

CircRNAs are single-stranded RNA molecules originating from a process of alternative splicing called backsplicing. CircRNAs have been implicated in several diseases including cancer, indeed some of them can be differentially expressed in cancer tissues and plasma, thus making them potential therapeutic targets or diagnostic biomarkers. Focusing on Non-Small Cell Lung Cancer (NSCLC), which is currently one of the main big-killer tumors due to its frequency and its fatal outcome, several circRNAs have been implicated in its progression and could be identified in patient plasma. Among them, circFARSA seems to have a role in the immune evasion of NSCLC. The most expressed circFARSA isoforms in NSCLC is derived from the circularization of 5-6-7 exons of FARSA gene. With this work, we aim at investigating circFARSA functional role in NSCLC cell lines by three main steps: i) investigation of potential circFARSA biological role in NSCLC cell lines ii) identification of potential circFARSA-derived peptide iii) prediction of circFARSA-RBP interaction. As a model, we selected three NSCLC cell lines: one adenocarcinoma (A549), one squamous carcinoma (NCI-H520), and one lymph node derived-metastatic cell line (NCI-H2030), all compared with a healthy bronchial-epithelium cell line (16HBE14o-). By qRT-PCR, we observed that circFARSA was differentially expressed among NSCLC cell lines and healthy control. Namely, in NCI-H520 NSCLC cell line a 17-fold circFARSA expression was observed if compared to the healthy 16HBE14o- control. Subsequently, to investigate the biological role of circFARSA in these cells, we design a specific circFARSA-siRNA recognizing the backsplicing junction and we verified that the silencing was specific for the circular counterpart without affecting the linear isoform expression in all cell lines. CircFARSA-silenced cells were essayed in viability and migration tests in comparison with mock-treated controls (unspecific control siRNA). The viability of NSCLC was significantly impaired after circFARSA silencing, if compared to control cells. However, the most impressive effect was observed on the migration assay: a 70% and a 40% of reduction of the migration was observed in A549 and NCI-H2030 respectively. These results showed that circFARSA could have a potential role in the cancer progression. CircRNA function is still a wide-open question. Exploring the circular sequence, a novel ORF was detected, encoding for a predicted 10.25 kDa in the same reading frame as the normal protein. The availability of an antibody recognizing this protein part was found, and immunoblotting analysis of cells demonstrated the presence of a protein that is sensitive to circFARSA downregulation using siRNA, albeit the protein has a higher molecular weight than expected. This point will require more intensive studies using polyribosome association assay as well as mass spectrometry studies. As a second step we examined whether circFARSA may act as a protein-sponge. An RBP suite database allowed the identification of IGF2BP3 as the best potential interactor for circFARSA (score 0.98). IGF2BP3 is an important m6A regulator that promotes the oncogene Myc targets V1 and mTORC1 survival signaling, so that we can speculate a potential involvement of this pathway in NSCLC proliferation, progression, and deserves further experimental assessments as a potential therapeutic target in NSCLC.