ИСТИНА

Neuroblastoma is the most common extracranial solid tumor of infancy, arising from the neuroblasts of the sympathetic nervous system (pluripotent sympathetic cells). These tumors can regress spontaneously or differentiate into a benign ganglioneuroma. However, in most of the cases neuroblastoma have extensive growth or metastatic spreading with a highly invasive cell phenotype and overall poor prognosis. The molecular basis of neuroblastoma progression is still obscure. Previously it was shown that the activation of urokinase system (urokinase (uPA) and its receptor uPAR) stimulates cell migration and invasion in many cancers. Here we show a new uPAR-dependent mechanism of the epithelial-mesenchymal transition (EMT) in murine Neuro 2A neuroblastoma cells. We employed CRISPR/Cas9 nickase system to target uPAR gene in Neuro 2A cells. The designed constructs effectively suppressed uPAR expression in the total population of transfected cells and in the selected clones after single cell plating. uPAR overexpression in Neuro 2A cells was carried out using plasmid transfection and antibiotic stable cell line establishment. uPAR suppression had a great impact on cell morphology: uPAR-deficient cells (Neuro2a-ΔuPAR) exhibited 4-folds (p<0. 05) increase in the cell size compared to the control cells. In a scratch wound assay, 12 hour uPA administration stimulated a 1. 5-fold increase (p<0. 05) in cell migration of Neuro 2A-ΔuPAR cells compared to the control and to uPAR-overexpressing cells (Neuro 2A-uPAR). After 24 hours Neuro2a-ΔuPAR cells almost completely (up to 79%, p<0. 05) recolonized the scratch wound, while control and Neuro 2A-uPAR cells recolonized it to a much lesser extent (9 and 12%, correspondingly). Moreover, control cells and cells overexpressing uPAR were characterized by a clonogenic growth type, while Neuro2a-ΔuPAR cells actively migrated and formed a uniform monolayer. uPAR deficiency was accompanied by a significant increase in IL-6 expression – a highly potent migratory and EMT inducing factor in neuroblastomas. These results suggest a novel mechanism of uPAR-dependent epithelial-mesenchymal transition in neuroblastoma cell.