Kryvdiuk I. ERN1-dependent regulation of GADD and TNF receptors gene expression in glioma cells

Object: mechanisms of ERN1-mediated human glioma cells proliferation. Aim: to investigate GADD and TNF receptors gene expression in U87 glioma cell line with ERN1 suppression in conditions of hypoxia and glutamine or glucose deprivation, in order to identify the possible role of these genes in ERN1-mediated growth control and proliferation of glioma cells. Methods: cell cultivation, RNA and proteins extraction, spectrophotometric assessment of RNA quantity and quality, cDNA synthesis, polymerase chain reaction, nucleic acid electrophoresis, restriction of plasmid DNA, methods of molecular cloning, liposome transfection of cells, Western blot analysis, statistical methods of data processing. For the first time it was shown that GADD and TNF receptors gene expression in glioma cells depended on ERN1 functional activity, and detected changes in their expression correlated with tumor growth inhibition of glioma cells with ERN1 blockade. It was also found that hypoxia reduced the level of TNFRSF11B, TNFRSF21 and FADD gene expressions and increased the level of TNFRSF10D, RYBP, TNIP1 genes and all studied members of GADD family, except SESN1 gene. Inhibition of ERN1 introduces hypoxia-induced sensitivity to TNFRSF1A, TNFRSF10B and SESN1 gene expressions. Also, it was shown that glutamine deprivation condition leads to up-regulation of all studied members of GADD family at the mRNA level in glioma cells, and this effect depends on the ERN1 functional activity. It was demonstrated that in control glioma cells glucose deprivation condition leads to the up-regulated expression of all studied members of GADD family, except SESN1, and the concomitant down-regulation of the majority of TNF receptors and related factors. The inhibition of ERN1 modifies the effect of glucose deprivation on the expression of most studied genes. Thus, these results demonstrate that fine-tuning of the expression of TNF-related factors, TNF receptor superfamily genes and GADD family genes is regulated by ERN1, an effector of endoplasmic reticulum stress, as well as depends on hypoxia, glucose and glutamine deprivation in gene specific manner.