Biotechnologia Acta (Apr 2022)
GLUTAMINE DEPRIVATION AFFECTS THE EXPRESSION OF GENES WHICH CONTROL PYRUVATE DEHYDROGENASE ACTIVITY: THE IMPACT OF ERN1 KNOCKDOWN
Abstract
The aim of the current investigation was to study the expression of genes encoded pyruvate dehydrogenase subunits (PDHA1, PDHB, PDHX, DLAT, and DLD) in U87 glioma cells in response to glutamine deprivation in U87 glioma cells in relation to knockdown of ERN1 for evaluation of a possible dependence of the expression of these important regulatory genes from glutamine supply and ERN1 signaling. Methods. The expression of PDHA1, PDHB, PDHX, DLAT, and DLD genes was studied by real-time qPCR in control U87 glioma cells (transfected by vector) and cells with knockdown of ERN1 (transfected by dnERN1) after exposure to glutamine deprivation condition. Total RNA was extracted from glioma cells using TRIZOL reagent. An RNA quantity as well as spectral characteristics was measured using NanoDrop One. For reverse transcription of mRNAs we used Thermo Scientific Verso cDNA Synthesis Kit (Germany). The values of mRNA expressions were normalized to the level of ACTB mRNA and represented as percent of control (100 %). Results. It was shown that the expression level of PDH1, PDHB, DLAT, and DLD genes was down-regulated in control glioma cells treated by glutamine deprivation. At the same time, ERN1 knockdown is suppressed the effect of glutamine deprivation on PDHB and DLD gene expressions in glioma cells, but did not change significantly the impact of glutamine deprivation on the expression of PDHA1, DLAT, and PDHX genes. Conclusions. The results of this investigation demonstrated that the expression of PDH1, PDHB, PDHX, DLAT, and DLD genes was significantly affected by exposure of U87 glioma cells under glutamine deprivation condition and that the effect of glutamine deprivation on the expression of most these genes was modified in cells with knockdown of ERN1, a major signaling pathway of the endoplasmic reticulum stress.
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