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

Abstract

Objective. 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.

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

“…The expression levels of IDE and PITRM1 mRNAs as well as ACTB mRNA were measured in control U87 glioma cells and cells with a deficiency of ERN1, introduced by dnERN1 and dnrERN1, by quantitative polymerase chain reaction using SYBRGreen Mix (ABgene, Thermo Fisher Scientific, Epsom, Surrey, UK) and "QuantStudio 5 Real-Time PCR System" (Applied Biosystems, USA). Thermo Scien tific Verso cDNA Synthesis Kit (Germany) was used for reverse transcription as described previously (Minchenko et al 2019). For polyadenylation and reverse transcription of miRNAs we used NCode™ miRNA First-Strand cDNA Synthesis Kit MIRC-10 (Invitrogen) as described previously (Minchenko et al 2018b).…”

Involvement of the VEGF Signaling Pathway in Immunosuppression and Hypoxia Stress: Analysis of mRNA Expression in Lymphocytes Mediating Panting in Jersey Cattle under Heat Stress

“…In In a previous study, hypoxia was found to affect cytokine gene expression, in ammation and immunosuppression in immune cells in a manner that involved VEGF, PTGS2, EDN1, CXCL8, IL1A and MYL9. Hypoxia has also been found to affect the expression of the gene encoding EDN1 in U87 glioma cells [11] and the genes encoding PTGS2 and VEGFA in breast cancer and melanoma cells [9]. Overproduction of PTGS2 has also been observed in lung cancer for immunosuppression [10].…”

“…For example, immune function is impaired in the early in ammation phase due to increased white blood cell counts when dairy cattle are under heat stress [8]. Furthermore, previous studies have reported that the effects of hypoxia on cytokine gene expression, in ammation and immunosuppression involve VEGF [9], PTGS2 [9][10], endothelin-1 (EDN1) [11], nuclear receptor subfamily 4 group A member 2 (NR4A2) [12][13][14], C-X-C motif chemokine ligand 8 (CXCL8) [15][16], interleukin 1 alpha (IL1A) [17] and MYL9 [18]. It is possible for heat to induce both hypoxia and immune responses to affect the expression of common genes with related functions.…”

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

“…The expression levels of homeobox mRNAs as well as ACTB mRNA were measured in control U87 glioma cells and cells with a deficiency of ERN1, introduced by dnERN1, by quantitative polymerase chain reaction using SYBRGreen Mix (ABgene, Thermo Fisher Scientific, Epsom, Surrey, UK) and "QuantStudio 5 Real-Time PCR System" (Applied Biosystems, USA). Thermo Scientific Verso cDNA Synthesis Kit (Germany) was used for reverse transcription as described previously (Minchenko et al 2019). Polymerase chain reaction was performed in triplicate.…”