Halkin O. Expression of protease genes in IRE1 knockdown U87 glioma cells.

Object: Molecular mechanisms of protease genes expression in signaling enzyme IRE1 knockdown U87 glioma cells, as well as upon hypoxia, glucose or glutamine deprivation in response to IRE1 activity. Methods: Western blot analysis, isolation of RNA and protein extracts from cells, nanospectrophotometric determination of RNA concentrations and spectral characteristics, complementary DNA synthesis by reverse transcriptase, real-time quantitative polymerase chain reaction, electrophoretic nucleic acid analysis, cell culture and statistical analysis of the results. The thesis highlights investigation of protease genes expression upon hypoxia and glucose or glutamine deprivation in IRE1 knockdown U87 glioma cells with suppressed kinase and endoribonuclease activities and their possible participation in IRE1-dependent growth of malignant tumors, in particular glioma. It was shown that inhibition of ІRE1 signaling enzyme activity in U87 glioma cells led to suppression of USP4, CTSC, CTSK and CTSS gene expressions, while for all other tested genes an increase of the expression level were observed, indicating the differential character of IRE1-dependent regulation of their expression. It has been established that the expression level of most of investigated specific for ubiquitin peptidase and cathepsin genes, as well as the HTRA1 and STC2 genes, are dependent from hypoxia, glutamate and glucose deprivation in IRE1-dependent manner. The changes in the expression of CTSC, CTSL, CTSS, HTRA1 and SCT2 genes in U87 glioma cells upon IRE1 inhibition are associated with reduced glioma cells proliferation and tumor growth and can be used for identification of mere efficient ways to suppression of glioma growth.