Garmash I. ERN1 regulation of genes expression encoded enzymes synthesis of glycolysis and pentose phosphate pathway.

Object: genes expression levels of GPI, GNPDA1, ENO1, ENO2, ALDOA, ALDOC, PRPS1, PRPS2, PRPSAP1, PRPSAP2, G6PD, PGLS, RPIA, TALDO1 and TKT of human. Aim: investigation of glycolysis and pentose phosphate pathway genes expression in glioma cell lines U87 under inhibition of ERN1 enzymatic activity, which is a main sensor enzyme of ER stress, in conditions of hypoxia and deprivation of glucose and glutamine. Methods: cultivation of glioma cell line U87 and its sublines, total RNA extraction from these cells, spectrophotometric determination of the nucleic acid concentration, cDNA synthesis by reverse transcription, polymerase chain reaction, quantitative polymerase chain reaction, electrophoretic analysis of amplification products, Western blot analysis of proteins (extraction of cytosol protein fractions from the cell cultures, spectrophotometric identification of protein amounts, protein polyacrylamide electrophoresis and immunoblotting), bioinformatic analysis, computer analysis of polymerase chain reaction results and statistical methods of data processing For the first time it have been demonstrated that inhibition of ERN1, which is key enzyme of ER stress signal pathway, leads to changes of genes expression levels of certain enzymes involved in glycolysis (GPI, ALDOA, ALDOC, ENO1 and ENO2), pentose phosphate pathway (G6PD, PGLS, RPIA, TKT and TALDO1) and associated with these metabolic pathways enzymes (GNPDA1, PRPS1, PRPS2, PRPSAP1 and PRPSAP2). The effect indicates an important role of these enzymes in malignant growth of glioma cells. It was found that changes in genes expression levels are mediated by both endoribonuclease and proteinkinase activities of ERN1 enzyme. It has been proved that ERN1 inhibition modifies effects of hypoxia and deprivation of nutrients (glucose and glutamine) on expression levels of certain genes. Obtained results will be used to develop fundamentally new approaches to the creation of anti-tumor drugs for the control of gliomas and in further scientific work.