Kharkova A. ERN1-dependent regulation of IGF system genes expression in glioma cells

Object: mechanisms of ERN1-mediated human glioma cells proliferation. Aim: to investigate the insulin-like growth factor system genes expression in glioma cell lines U87 with inhibition of sensor and signaling endoplasmic reticulum stress enzyme ERN1 function in order to identify the possible role of these genes in ERN1-mediated proliferation of glioma cells. Methods: glioma cell line U87 and its sublines cultivation, total RNA extraction, agarose electrophoresis of nucleic acids, reverse transcription, polymerase chain reaction, quantitative polymerase chain reaction, extraction of cytosol and nucleus protein fractions from the cell cultures, precipitation of proteins from the cell culture media, spectrophotometric identification of protein amounts, protein polyacrylamide electrophoresis, immunoblotting, computer analysis of polymerase chain reaction results, statistical methods of data processing. It was first shown that the expression of IGF1, IGF2, IGF1R, IRS1, IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6 and IGF2BP3 genes depends on ERN1 functional activity and changes in their expression correlates with suppression of proliferation of glioma cell with ERN1 blockade. We also established that the expression of pro-proliferative genes was reduced in glioma cells with suppressed function of ERN1. At the same time, the level of gene expression of anti-proliferative factors was increased in cells with ERN1 blockade. It was also shown that hypoxia, as well as glucose and glutamine deprivation influenced the expression of pro- and anti-proliferative genes expression.