PO-220 RAN, a novel and promising gene for malignant pleural mesothelioma

Introduction RAN is a member of RAS superfamily of GTPases involved in a varied range of cellular processes. Although it is widely demonstrated RAN is overexpressed in many human tumours having an essential role in malignant cell survival and cancer progression, little is known about its role in Malignant Pleural Mesothelioma (MPM). Previous studies showed the RAN gene is upregulated in mesothelioma tissues and cell lines, so it might be involved in carcinogenesis of MPM. We aimed to explore the functional role of RAN in MPM cell lines and its likely use as co-target in mesothelioma treatment. Material and methods The role of RAN in MPM tumorigenesis was investigated through RNA interference, on a panel of one mesothelial cell line (Met-5A) and four MPM cell lines (Mero-14, Mero-25, Istmes-2 and NCI-H28). After monitoring gene knockdown, at both the mRNA and protein levels, a phenotypic study was performed through Caspase-3/7, Sulforhodamine B, Wound-Healing and Colony Formation assays. Flow cytometry was employed to monitor cell cycle. To validate data from siRNA experiments, two different siRNA were independently used to target RAN. The gene was also knocked-out using a lentiviral CRISPR/Cas9 system in Mero-14. Cas9 endonuclease and gRNA were transduced by two different lentiviral transfer vectors.The doxycycline-regulated Cas9 induction was followed by DNA, RNA and proteins extraction to confirm the occurrence of gene disruption. TIDE analysis was carried out to monitor targeted mutations triggered by the genome editing. Results and discussions The siRNA-mediated knockdown was confirmed at both the mRNA and protein level in all cell lines. The silencing caused a statistically significant decrease of proliferation rate and clonogenicity in Mero-14, Mero-25 and Istmes-2.The migration ability was affected in Met-5A and Istmes-2. An increase in apoptosis was observed in all cell lines, being statistically significant only in the malignant ones. Flow cytometry analysis showed an increase of cells in G0/G1 phase and a decrease of cells in S phase, being significant in Mero-14 cell line only. RAN knock-out has been confirmed at both the mRNA and protein level, whereas the TIDE analysis is still ongoing. Conclusion This study showed that MFAP5 is a novel myoepithelial cell marker that appears to be up-regulated in duct epithelium in DCIS and IC-NST during tumourogenesis and that its cytoplasmic expression in invasive tumours seems to have apoor prognostic role manifested by its association with poor prognostic parameters such as high grade, late stage,lymph node invasion and increased MVD.