scholarly journals Expression of IDE and PITRM1 genes in ERN1 knockdown U87 glioma cells: effect of hypoxia and glucose deprivation

2020 ◽  
Vol 54 (3) ◽  
pp. 183-195
Author(s):  
Dmytro O. Minchenko ◽  
Olena O. Khita ◽  
Dariia O. Tsymbal ◽  
Serhij V. Danilovskyi ◽  
Olha V. Rudnytska ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Kinase ◽  
Endoplasmic Reticulum Stress ◽  
Specific Binding ◽  
Quantitative Polymerase Chain Reaction ◽  
Glioma Cells ◽  
Glucose Deprivation ◽  
Expression Level ◽  
Insulin Degrading Enzyme ◽  
Gene Expressions

AbstractObjective. The aim of the present investigation was to study the expression of genes encoding polyfunctional proteins insulinase (insulin degrading enzyme, IDE) and pitrilysin metallopeptidase 1 (PITRM1) in U87 glioma cells in response to inhibition of endoplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) for evaluation of their possible significance in the control of metabolism through ERN1 signaling as well as hypoxia, glucose and glutamine deprivations.Methods. The expression level of IDE and PITRM1 genes was studied in control and ERN1 knockdown U87 glioma cells under glucose and glutamine deprivations as well as hypoxia by quantitative polymerase chain reaction.Results. It was found that the expression level of IDE and PITRM1 genes was down-regulated in ERN1 knockdown (without ERN1 protein kinase and endoribonuclease activity) glioma cells in comparison with the control glioma cells, being more significant for PITRM1 gene. We also found up-regulation of microRNA MIR7-2 and MIRLET7A2, which have specific binding sites in 3’-untranslated region of IDE and PITRM1 mRNAs, correspondingly, and can participate in posttranscriptional regulation of these mRNA expressions. Only inhibition of ERN1 endoribonuclease did not change significantly the expression of IDE and PITRM1 genes in glioma cells. The expression of IDE and PITRM1 genes is preferentially regulated by ERN1 protein kinase. We also showed that hypoxia down-regulated the expression of IDE and PITRM1 genes and that knockdown of ERN1 signaling enzyme function modified the response of these gene expressions to hypoxia. Glucose deprivation increased the expression level of IDE and PITRM1 genes, but ERN1 knockdown enhanced only the effect of glucose deprivation on PITRM1 gene expression. Glutamine deprivation did not affect the expression of IDE gene in both types of glioma cells, but up-regulated PITRM1 gene and this up-regulation was stronger in ERN1 knockdown cells.Conclusions. Results of this investigation demonstrate that ERN1 knockdown significantly decreases the expression of IDE and PITRM1 genes by ERN1 protein kinase mediated mechanism. The expression of both studied genes was sensitive to hypoxia as well as glucose deprivation and dependent on ERN1 signaling in gene-specific manner. It is possible that the level of these genes expression under hypoxia and glucose deprivation is a result of complex interaction of variable endoplasmic reticulum stress related and unrelated regulatory factors and contributed to the control of the cell metabolism.

scholarly journals ERN1 knockdown modifies the effect of glucose deprivation on homeobox gene expressions in U87 glioma cells

2020 ◽  
Vol 54 (3) ◽  
pp. 196-206
Author(s):  
Dariia O. Tsymbal ◽  
Dmytro O. Minchenko ◽  
Olena O. Khita ◽  
Olha V. Rudnytska ◽  
Yulia M. Viletska ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Quantitative Polymerase Chain Reaction ◽  
Homeobox Gene ◽  
Glioma Cells ◽  
Glucose Deprivation ◽  
Expression Level ◽  
Deprivation Condition ◽  
Gene Expressions ◽  
Major Pathway

AbstractObjective. The aim of the present investigation was to study the expression of genes encoding homeobox proteins ZEB2 (zinc finger E-box binding homeobox 2), TGIF1 (TGFB induced factor homeobox 1), SPAG4 (sperm associated antigen 4), LHX1 (LIM homeobox 1), LHX2, LHX6, NKX3-1 (NK3 homeobox 1), and PRRX1 (paired related homeobox 1) in U87 glioma cells in response to glucose deprivation in control glioma cells and cells with knockdown of ERN1 (endoplasmic reticulum to nucleus signaling 1), the major pathway of the endoplasmic reticulum stress signaling, for evaluation of it possible significance in the control of glioma growth through ERN1 signaling and chemoresistance.Methods. The expression level of homeobox family genes was studied in control (transfected by vector) and ERN1 knockdown U87 glioma cells under glucose deprivation condition by real-time quantitative polymerase chain reaction.Results. It was shown that the expression level of ZEB2, TGIF1, PRRX1, and LHX6 genes was up-regulated in control glioma cells treated by glucose deprivation. At the same time, the expression level of three other genes (NKX3-1, LHX1, and LHX2) was down-regulated. Furthermore, ERN1 knockdown of glioma cells significantly modified the effect glucose deprivation condition on the expression almost all studied genes. Thus, treatment of glioma cells without ERN1 enzymatic activity by glucose deprivation condition lead to down-regulation of the expression level of ZEB2 and SPAG4 as well as to more significant up-regulation of PRRX1 and TGIF1 genes. Moreover, the expression of LHX6 and NKX3-1 genes lost their sensitivity to glucose deprivation but LHX1 and LHX2 genes did not change it significantly.Conclusions. The results of this investigation demonstrate that ERN1 knockdown significantly modifies the sensitivity of most studied homeobox gene expressions to glucose deprivation condition and that these changes are a result of complex interaction of variable endoplasmic reticulum stress related and unrelated regulatory factors and contributed to glioma cell growth and possibly to their chemoresistance.

scholarly journals Glucose deprivation affects the expression of genes encoding cAMP-activated protein kinase and related proteins in U87 glioma cells in ERN1 dependent manner

2020 ◽  
Vol 54 (4) ◽  
pp. 244-254
Author(s):  
Oksana O. Ratushna
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Kinase ◽  
Endoplasmic Reticulum Stress ◽  
Glioma Cells ◽  
Glucose Deprivation ◽  
Expression Level ◽  
Gene Expressions ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Related Proteins

Abstract Objective. The aim of this investigation was to study the expression of genes encoding cAMP-activated protein kinase catalytic and regulatory A subunits (PRKACA and PRKAR1A) and related proteins such as cAMP-dependent protein kinase inhibitors A and G (PKIA and PKIG), catalytic subunit A of protein phosphatase 3 (PPP3CA), A-kinase anchoring protein 12 (AKAP12), and praja ring finger ubiquitin ligase 2 (PJA2) in U87 glioma cells in response to glucose deprivation in both control U87 glioma cells and cells with ERN1 (endoplasmic reticulum to nucleus signaling 1) knockdown, the major pathway of the endoplasmic reticulum stress signaling, for evaluation of possible significance of glucose deprivation in ERN1 dependent regulation of glioma growth. Methods. The expression level of PRKA related genes was studied in control (transfected by vector) and ERN1 knockdown U87 glioma cells under glucose deprivation by real-time quantitative polymerase chain reaction. Results. It was shown that the expression level of PRKACA and PKIA genes was down-regulated in control glioma cells treated by glucose deprivation, but PJA2 gene was up-regulated. At the same time, the expression of four other genes (PRKAR1A, PKIG, AKAP12, and PPP3CA) was resistant to this experimental condition. Furthermore, ERN1 knockdown of glioma cells significantly modified the effect glucose deprivation on the expression almost all studied genes. Thus, treatment of glioma cells with inhibited ERN1 enzymatic activity by glucose deprivation lead to a more significant down-regulation of the expression level of PKIA and to suppression PRKAR1A gene expressions. Moreover, the ERN1 knockdown introduced up-regulation of PKIG and AKAP12 gene expressions in glioma cells treated by glucose deprivation and eliminated the sensitivity of PJA2 gene to this experimental condition. Conclusions. Results of this investigation demonstrated that ERN1 knockdown significantly modified the sensitivity of most studied PRKA related gene expressions to glucose deprivation and that these changes are a result of complex interactions of variable endoplasmic reticulum stress related and unrelated regulatory factors and contributed to the suppression of glioma cell proliferation and their possibly chemoresistance.

scholarly journals Hypoxic regulation of EDN1, EDNRA, EDNRB, and ECE1 gene expressions in ERN1 knockdown U87 glioma cells

2019 ◽  
Vol 53 (4) ◽  
pp. 250-262 ◽  
Author(s):  
Dmytro O. Minchenko ◽  
Daria O. Tsymbal ◽  
Olena O. Riabovol ◽  
Yuliia M. Viletska ◽  
Yuliia O. Lahanovska ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Specific Binding ◽  
Quantitative Polymerase Chain Reaction ◽  
Glioma Cells ◽  
Micro Rna ◽  
Expression Level ◽  
Gene Expressions ◽  
Down Regulation ◽  
Endothelin 1 ◽  
Glioma Growth

AbstractObjective. The aim of the present investigation was to study the effect of hypoxia on the expression of genes encoding endothelin-1 (EDN1) and its cognate receptors (EDNRA and EDNRB) as well as endothelin converting enzyme 1 (ECE1) in U87 glioma cells in response to inhibition of endoplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) for evaluation of their possible significance in the control of glioma growth through ERN1 and hypoxia.Methods. The expression level of EDN1, EDNRA, EDNRB, and ECE1 genes as well as micro-RNA miR-19, miR-96, and miR-206 was studied in control and ERN1 knockdown U87 glioma cells under hypoxia by quantitative polymerase chain reaction.Results. It was shown that the expression level of EDN1, EDNRA, EDNRB, and ECE1 genes was up-regulated in ERN1 knockdown glioma cells in comparison with the control glioma cells, being more significant for endothelin-1. We also observed down-regulation of microRNA miR-206, miR-96, and miR-19a, which have specific binding sites in mRNA EDN1, EDNRA, and EDNRB, correspondingly, and can participate in posttranscriptional regulation of these mRNA expressions. Furthermore, inhibition of ERN1 endoribonuclease lead to up-regulation of EDNRA and ECE1 gene expressions and down-regulation of the expression level of EDN1 and EDNRB genes in glioma cells. Thus, the expression of EDNRA and ECE1 genes is regulated by ERN1 endoribonuclease, but EDN1 and EDNRB genes preferentially by ERN1 protein kinase. We have also shown that hypoxia enhanced the expression of EDN1, EDNRA, and ECE1 genes and that knockdown of ERN1 signaling enzyme function significantly modified the response of all studied gene expressions to hypoxia. Thus, effect of hypoxia on the expression level of EDN1 and ECE1 genes was significantly or completely reduced in ERN1 knockdown glioma cells since the expression of EDNRA gene was down-regulated under hypoxia. Moreover, hypoxia is induced the expression of EDNRB gene in ERN1 knockdown glioma cells.Conclusions. Results of this investigation demonstrate that ERN1 knockdown significantly increased the expression of endothelin-1 and its receptors as well as ECE1 genes by different mechanisms and that all studied gene expressions were sensitive to hypoxia. It is possible that hypoxic regulation of the expression of these genes is a result of complex interaction of variable ERN1 related transcription and regulatory factors with HIF1A and possibly contributed to the control of glioma growth.

scholarly journals Hypoxic regulation of the expression of genes encoded estrogen related proteins in U87 glioma cells: eff ect of IRE1 inhibition

2017 ◽  
Vol 51 (1) ◽  
pp. 8-19 ◽  
Author(s):  
Minchenko Do ◽  
Riabovol Oo ◽  
Ratushna Oo ◽  
Minchenko Oh
Keyword(s):  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Quantitative Polymerase Chain Reaction ◽  
Glioma Cells ◽  
Hypoxic Condition ◽  
Enzyme Function ◽  
Unfolded Protein ◽  
Expression Of Genes ◽  
The Unfolded Protein Response ◽  
Related Proteins

Abstract Objective. The aim of the present study was to examine the effect of inhibition of endoplasmic reticulum stress signaling, mediated by IRE1 (inositol requiring enzyme 1), which is a central mediator of the unfolded protein response on the expression of genes encoded estrogen related proteins (NRIP1/RIP140, TRIM16/EBBP, ESRRA/NR3B1, FAM162A/E2IG5, PGRMC2/PMBP, and SLC39A6/LIV-1) and their hypoxic regulation in U87 glioma cells for evaluation of their possible significance in the control of glioma cells proliferation.Methods. The expression of NRIP1, EBBP, ESRRA, E2IG5, PGRMC2, and SLC39A6 genes in U87 glioma cells, transfected by empty vector pcDNA3.1 (control) and cells without IRE1 signaling enzyme function (transfected by dnIRE1) upon hypoxia, was studied by a quantitative polymerase chain reaction.Results. Inhibition of both enzymatic activities (kinase and endoribonuclease) of IRE1 signaling enzyme function up-regulates the expression of EBBP, E2IG5, PGRMC2, and SLC39A6 genes is in U87 glioma cells in comparison with the control glioma cells, with more significant changes for E2IG5 and PGRMC2 genes. At the same time, the expression of NRIP1 and ESRRA genes is strongly down-regulated in glioma cells upon inhibition of IRE1. We also showed that hypoxia increases the expression of E2IG5, PGRMC2, and EBBP genes and decreases NRIP1 and ESRRA genes expression in control glioma cells. Furthermore, the inhibition of IRE1 in U87 glioma cells decreases the eff ect of hypoxia on the expression of E2IG5 and PGRMC2 genes, eliminates hypoxic regulation of NRIP1 gene, and enhances the sensitivity of ESRRA gene to hypoxic condition. Furthermore, the expression of SLC39A6 gene is resistant to hypoxia in both the glioma cells with and without IRE1 signaling enzyme function.Conclusions. Results of this investigation demonstrate that inhibition of IRE1 signaling enzyme function affects the expression of NRIP1, EBBP, ESRRA, E2IG5, PGRMC2, and SLC39A6 genes in U87 glioma cells in gene specific manner and these changes possibly contribute to the suppression of the cell proliferation. Most of these genes are regulated by hypoxia and preferentially through IRE1 signaling pathway of endoplasmic reticulum stress.

scholarly journals Factors and their receptor genes in glioma cells with suppressed function of ERN 1 enzyme upon glucose deprivation

2016 ◽  
Vol 20 (1) ◽  
pp. 44-49
Author(s):  
A. Kharkova ◽  
O. Minchenko
Keyword(s):  
Gene Expression ◽  
Glioma Cells ◽  
Glucose Deprivation ◽  
Expression Level ◽  
Enzyme Function ◽  
Gene Expressions ◽  
Igf2 Gene ◽  
Igf Receptor ◽  
Effect Of Glucose ◽  
Insulin Like Growth Factors

It was shown that the expression level of insulin like growth factors (IGF1 and IGF2) genes is decreased, but IGF receptor (IRF1R) gene is significantly increased in U87 glioma cells with suppressed activity of the sensor and signaling enzyme ERN1. In U87 glioma cells the expression level of IGF1 gene is decreased but IGF2 and IGF1R do not change significantly upon glucose deprivation condition. The inhibition of ERN1 functional activity does not affect the sensitivity of IGF1 and IGF1R gene expressions to glucose deprivation but the inhibition of ERN1 eliminates the effect of glucose deprivation on IGF2 gene expression. Thus, the IGF1, IGF2 and IGF1R genes are related to the regulation of glioma cells proliferation and are sensitive to glucose deprivation in dependence of ERN1 enzyme function.

scholarly journals Expression of IGFBP1, IGFBP2 and IGF2BP3 genes in U87 glioma cells with suppressed ERN1 signaling enzyme function in glutamine and glucose deprivation conditions

2014 ◽  
Vol 68 (3) ◽  
pp. 24-29
Author(s):  
A. Kharkova ◽  
D. Minchenko ◽  
D. Tsymbal ◽  
O. Minchenko
Keyword(s):  
Cell Proliferation ◽  
Endoplasmic Reticulum ◽  
Glioma Cells ◽  
Glucose Deprivation ◽  
Enzyme Function ◽  
Deprivation Condition ◽  
Loss Of Function ◽  
Gene Expressions ◽  
Glutamine Deprivation ◽  
Effect Of Glucose

Insulin-like growth factor binding proteins play an important role in the regulation of cell proliferation and malignant tumor growth. It was shown that blockade of both enzymatic functions of sensor and signaling enzyme ERN1 (from endoplasmic reticulum to nuclei-1), the major component of endoplasmic reticulum stress signaling, decreases the expression level of IGFBP1, IGFBP2 and IGF2BP3 genes in U87 glioma cell. The decreased level of these gene expressions in glioma cells with ERN1 signaling enzyme loss of function correlates with suppression of cell proliferation. It was shown that glutamine deprivation condition leads to enhance the expression of IGFBP1 gene, but did not change significantly the expression of IGFBP2 and IGF2BP3 genes in both types of glioma cells. Moreover, this effect of glutamine deprivation did not depend from suppression of ERN1 enzyme function. At the same time, the expression of IGFBP2 and IGF2BP3 genes is decreased in glucose deprivation condition in both types of glioma cells and blockade of ERN1 signaling enzyme enhanced this effect. Thus, results of this investigation demonstrated that the expression of IGFBP1, IGFBP2 and IGF2BP3 genes in U87 glioma cells is dependent from signaling enzyme ERN1 and is changed in glutamine and glucose deprivation conditions, but only effect of glucose deprivation was depended of ERN1 signaling enzyme function. Moreover, the decreasing of IGFBP1, IGFBP2 and IGF2BP3 gene expressions in glioma cells with blockade of both enzymatic activities of ERN1 is possibly related to suppression of these cells proliferation.

scholarly journals Inhibition of IRE1 signaling affects expression of a subset genes encoding for TNF-related factors and receptors and modifies their hypoxic regulation in U87 glioma cells

2016 ◽  
Vol 3 (1) ◽  
Author(s):  
Oleksandr H. Minchenko ◽  
Iryna V. Kryvdiuk ◽  
Dmytro O. Minchenko ◽  
Olena O. Riabovol ◽  
Oleh V. Halkin
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Death ◽  
Endoplasmic Reticulum Stress ◽  
Tumor Growth ◽  
Glioma Cells ◽  
Dominant Negative ◽  
Gene Expressions ◽  
Related Factors ◽  
Genes Encoding ◽  
Glioma Growth

AbstractInhibition of IRE1 (inositol requiring enzyme-1), the major signaling pathway of endoplasmic reticulum stress, significantly decreases tumor growth and proliferation of glioma cells. To elucidate the role of IRE1- mediated glioma growth, we studied the expression of a subset genes encoding for TNF (tumor necrosis factor)- related factors and receptors and their hypoxic regulation in U87 glioma cells overexpressing dominant-negative IRE1 (dnIRE1). We demonstrated that the expression of TNFAIP1, TNFRSF10D, TNFRSF21, TNFRSF11B, TNFSF7, and LITAF genes is increased in glioma cells with modified IRE1; however, TNFRSF10B, TRADD, and TNFAIP3 is down-regulated in these cells as compared to their control counterparts. We did not find TNFRSF1A gene expression to change significantly under this experimental condition. In control glioma cells, hypoxia leads to the up-regulated expression of TNFAIP1, TNFAIP3, TRADD, and TNFRSF10D genes and the concomitant down-regulation of TNFRSF21, TNFRSF11B, and LITAF genes; while, TNFRSF10B and TNFRSF1A genes are resistant to hypoxic treatment. However, inhibition of IRE1 modifies the hypoxic regulation of LITAF, TNFRSF21, TNFRSF11B, and TRADD genes and introduces hypoxia-induced sensitivity to TNFRSF10B, TNFRSF1A, and TNFSF7 gene expressions. Furthermore, knockdown by siRNA of TNFRSF21 mRNA modifies the hypoxic effect on the IRE1-dependent rate of proliferation and cell death in U87 glioma cells. The present study demonstrates that fine-tuned manipulation of the expression of TNF-related factors and receptors directly relating to cell death and proliferation, is mediated by an effector of endoplasmic reticulum stress, IRE1, as well as by hypoxia in a gene-specific manner. Thus, inhibition of the kinase and endoribonuclease activities of IRE1 correlates with deregulation of TNF-related factors and receptors in a manner that is gene specific and thus slows tumor growth.

scholarly journals Inhibition of kinase and endoribonuclease activity of ERN1/IRE1α affects expression of proliferationrelated genes in U87 glioma cells

2015 ◽  
Vol 2 (1) ◽  
Author(s):  
Oleksandr H. Minchenko ◽  
Dariia O. Tsymbal ◽  
Dmytro O. Minchenko ◽  
Michel Moenner ◽  
Olena V. Kovalevska ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Transcription Factor ◽  
Endoplasmic Reticulum Stress ◽  
Tumor Growth ◽  
Mrna Level ◽  
Glioma Cells ◽  
Dominant Negative ◽  
Fine Tuning ◽  
Gene Expressions ◽  
Endoribonuclease Activity

AbstractInhibition of ERN1/IRE1α (endoplasmic reticulum to nucleus signaling 1/inositol requiring enzyme-1α), the major signaling pathway of endoplasmic reticulum stress, significantly decreases tumor growth. We have studied the expression of transcription factors such as E2F8 (E2F transcription factor 8), EPAS1 (endothelial PAS domain protein 1), TBX3 (T-box 3), ATF3 (activating transcription factor 3), FOXF1 (forkhead box F1), and HOXC6 (homeobox C6) in U87 glioma cells overexpressing dominant-negative ERN1/IRE1α defective in endoribonuclease (dnr-ERN1) as well as defective in both kinase and endonuclease (dn-ERN1) activity of ERN1/IRE1α. We have demonstrated that the expression of all studied genes is decreased at the mRNA level in cells with modified ERN1/IRE1α; TBX3, however, is increased in these cells as compared to control glioma cells. Changes in protein levels of E2F8, HOXC6, ATF3, and TBX3 corresponded to changes in mRNAs levels. We also found that two mutated ERN1/IRE1α have differential effects on the expression of studied transcripts. The presence of kinase and endonuclease deficient ERN1/IRE1α in glioma cells had a less profound effect on the expression of E2F8, HOXC6, and TBX3 genes than the blockade of the endoribonuclease activity of ERN1/IRE1α alone. Kinase and endonuclease deficient ERN1/IRE1α suppresses ATF3 and FOXF1 gene expressions, while inhibition of only endoribonuclease of ERN1/IRE1α leads to the up-regulation of these gene transcripts. The present study demonstrates that fine-tuning of the expression of proliferation related genes is regulated by ERN1/IRE1α an effector of endoplasmic reticulum stress. Inhibition of ERN1/IRE1α, especially its endoribonuclease activity, correlates with deregulation of proliferation related genes and thus slower tumor growth.

scholarly journals Effect of glucose deprivation on the expression of genes encoding glucocorticoid receptor and some related factors in ERN1-knockdown U87 glioma cells

2019 ◽  
Vol 53 (4) ◽  
pp. 237-249 ◽  
Author(s):  
Olena O. Riabovol ◽  
Dariia O. Tsymbal ◽  
Dmytro O. Minchenko ◽  
Kateryna M. Lebid-Biletska ◽  
Myroslava Y. Sliusar ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Glioma Cells ◽  
Glucose Deprivation ◽  
Expression Level ◽  
Enzyme Function ◽  
Deprivation Condition ◽  
Stress Signaling ◽  
Expression Of Genes ◽  
Glioma Growth ◽  
Effect Of Glucose

AbstractObjective. The aim of the present study was to examine the effect of glucose deprivation on the expression of genes encoded glucocorticoid receptor (NR3C1) and some related proteins (NR3C2, AHR, NRIP1, NNT, ARHGAP35, SGK1, and SGK3) in U87 glioma cells in response to inhibition of endoplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1/inositol requiring enzyme 1) for evaluation of their possible significance in the control of glioma growth through endoplasmic reticulum stress signaling mediated by IRE1 and glucose deprivation.Methods. The expression of NR3C1, NR3C2, AHR, NRIP1, NNT, ARHGAP35, SGK1, and SGK3 genes in U87 glioma cells transfected by empty vector pcDNA3.1 (control cells) and cells without ERN1 signaling enzyme function (transfected by dnERN1) under glucose deprivation was studied by real time quantitative polymerase chain reaction.Results. It was shown that the expression level of NR3C2, AHR, SGK1, SGK3, and NNT genes was up-regulated in control U87 glioma cells under glucose deprivation condition in comparison with the control cells growing with glucose. At the same time, the expression of NRIP1 gene is down-regulated in these glioma cells under glucose deprivation, but NR3C1 and ARHGAP35 genes was resistant to this experimental condition. We also showed that inhibition of ERN1 signaling enzyme function significantly modified the response of most studied gene expressions to glucose deprivation condition. Thus, effect of glucose deprivation on the expression level of NR3C2, AHR, and SGK1 genes was significantly stronger in ERN1 knockdown U87 glioma cells since the expression of NNT gene was resistant to glucose deprivation condition. Moreover, the inhibition of ERN1 enzymatic activities in U87 glioma cells led to up-regulation of ARHGAP35 gene expression and significant down-regulation of the expression of SGK3 gene in response to glucose deprivation condition.Conclusions. Results of this study demonstrated that glucose deprivation did not change the expression level of NR3C1 gene but it significantly affected the expression of NR3C2, AHR, NRIP, SGK1, SGK3, and NNT genes in vector-transfected U87 glioma cells in gene specific manner and possibly contributed to the control of glioma growth since the expression of most studied genes in glucose deprivation condition was significantly dependent on the functional activity of IRE1 signaling enzyme.

scholarly journals Inhibition of IRE1 signaling affects the expression of genes encoded glucocorticoid receptor and some related factors and their hypoxic regulation in U87 glioma cells

2016 ◽  
Vol 50 (3) ◽  
pp. 127-136 ◽  
Author(s):  
D.O. Minchenko ◽  
O.O. Riabovol ◽  
D.O. Tsymbal ◽  
O.O. Ratushna ◽  
O.H. Minchenko
Keyword(s):  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Glucocorticoid Receptor ◽  
Quantitative Polymerase Chain Reaction ◽  
Glioma Cells ◽  
Hypoxic Condition ◽  
Enzyme Function ◽  
Related Factors ◽  
Expression Of Genes ◽  
Glioma Growth

Abstract Objective. The aim of the present investigation was to examine the effect of inhibition of endoplasmic reticulum stress signaling, mediated by IRE1 (inositol requiring enzyme 1), which is a central mediator of the unfolded protein response on the expression of genes encoding glucocorticoid receptor (NR3C1) and some related proteins (SGK1, SGK3, NCOA1, NCOA2, ARHGAP35, NNT) and their hypoxic regulation in U87 glioma cells for evaluation of their possible significance in the control of the glioma growth. Methods. The expression of NR3C1,SGK1,SGK3, NCOA1, NCOA2, ARHGAP35, and NNT genes in U87 glioma cells, transfected by empty vector pcDNA3.1 (control) and cells without IRE1 signaling enzyme function (transfected by dnIRE1) upon hypoxia, was studied by quantitative polymerase chain reaction. Results. Inhibition of IRE1 signaling enzyme function up-regulates the expression of NR3C1, SGK1, NCOA1, NCOA2, ARHGAP35, and NNT genes in U87 glioma cells in comparison with the control glioma cells, with more significant changes for NR3C1, SGK1, and NNT genes. At the same time, the expression of SGK3 gene is strongly down-regulated in glioma cells upon inhibition of IRE1. We have also shown that hypoxia increases the expression of NR3C1, SGK1, NCOA2, ARHGAP35, and NNT genes but decreases SGK3 and NCOA1 genes expression in control glioma cells. Moreover, the inhibition of both enzymatic activities (kinase and endoribonuclease) of IRE1 in U87 glioma cells enhances the eff ect of hypoxia on the expression of SGK1, SGK3, and NNT genes, but decreases the sensitivity of NR3C1 gene to hypoxic condition. Furthermore, the expression of NCOA1 gene is resistant to hypoxia in control glioma cells, but NCOA2 and ARHGAP35 genes are resistant to this condition in glioma cells without functional activity of IRE1 signaling enzyme. Conclusions. Results of this investigation demonstrate that inhibition of IRE1 signaling enzyme function affects the expression of NR3C1, SGK1, SGK3, NCOA1, NCOA2, ARHGAP35, and NNT genes in U87 glioma cells in gene specific manner and that all these genes are regulated by hypoxia preferentially through IRE1 signaling pathway of the endoplasmic reticulum stress.

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