Slіusar M. Molecular mechanisms of the serine synthesis gene regulation in glioma cells

The object of the study was the molecular mechanisms of regulation of the expression of serine synthesis genes under conditions of inhibition of ERN1 activity, as well as under hypoxia, glutamine, and glucose deficiency. The work aims to investigate the expression of serine synthesis genes in normal astrocytes and glioblastoma cells with suppressed ERN1 activity and under the influence of hypoxia and glutamine and glucose deficiency. Modern biochemistry and molecular biology methods, quantitative PCR, silencing, Western blot, and genetically modified glioblastoma cells were used. For the first time, the dependence of the expression level of serine synthesis genes on the inhibition of the ERN1 signaling protein was revealed, and changes in their expression for most genes were different in cells without both enzymatic activities of ERN1 and cells without its endoribonuclease activity of ERN1, which indicates different mechanisms of their regulation. A decrease in the expression of serine synthesis genes was found in cells with suppressed ERN1 functional activity, which is in good agreement with their reduced proliferative potential since increased expression of serine synthesis enzymes is an important condition for enhanced growth of malignant tumors. The results obtained demonstrated the important role of the endoribonuclease ERN1 in regulating ATF4 gene expression since the expression level of this gene did not differ significantly in magnitude in both glioma cells with suppressed endoribonuclease activity of the signaling protein ERN1 and in cells without both enzymatic activities of ERN1. It was shown for the first time that the protein kinase activity of ERN1 is an important regulator of the expression of the PHGDH, SHMT1, and SHMT2 genes since inhibition of the endoribonuclease activity of this signaling protein did not significantly affect their expression level, and only under conditions of inhibition of the protein kinase and endoribonuclease activities of the ERN1 protein did the expression level of these genes change. For the first time, it was established that the expression of all the studied genes is sensitive to hypoxia in glioblastoma cells, but changed differently in both the magnitude of the effect and the direction of the changes. It was found that inhibition of both enzymatic activities of the signaling protein ERN1 changes the sensitivity of the PHGDH, PSAT1, PSPH, and ATF4 genes to hypoxia, which indicates ERN1-dependent control of hypoxic regulation of the expression level of these genes. These results provide the basis for revealing the molecular mechanisms of glioblastoma cell resistance to hypoxia toxicity under endoplasmic reticulum stress. The practical significance of these results lies in elucidating the role of protein kinase and endoribonuclease ERN1 in decreasing the expression of serine synthesis genes and inhibiting glioma cell proliferation. The identified miRNAs that control the expression of PSAT1, PSPH, and SHMT1 mRNAs at the post-translational level may be potential targets for inhibiting glioblastoma cell proliferation. The molecular mechanisms we have identified for the resistance of glioblastoma cells to the toxic effects of hypoxia during endoplasmic reticulum stress are important for cancer treatment. The results of this study are included in the working program of the academic discipline: "Modern scientific approaches to biochemistry and biotechnology" for higher education applicants for the degree of Doctor of Philosophy (third educational and scientific level) in the specialty 091 Biology and Biochemistry.