Effect of hypoxia and glutamine or glucose deprivation on the expression of retinoblastoma and retinoblastoma-related genes in ERN1 knockdown glioma U87 cell line

Abstract

The expression of retinoblastoma and several retinoblastoma-related genes was studied in glioma cell line U87 and its subline with knockdown of ERN1 (endoplasmic reticulum-nuclei-1), the main endoplasmic reticulum stress sensing and signaling enzyme. It was shown that a blockade of the ERN1 enzyme function increases the expression levels of retinoblastoma, retinoblastoma-like 1 and most retinoblastoma related genes: EID1, JARID1B, E2F1, E2F3, RBAP48 and CTIP, does not change RNF40 and RBAP46 and decreases KDM5A. We have also demonstrated that hypoxia reduces the expression levels of retinoblastoma, EID1, and E2F1 in ERN1-deficient glioma cells only. At the same time, the expression levels of retinoblastoma-like 1, E2F3, RBAP46, RBAP48 and CTIP decrease, while JARID1B and RBBP2 increase in both types of cells in hypoxic conditions, but the expression is much stronger in cells with suppressed function of ERN1. The expression level of JARID1B and KDM-5A mRNA is also enhanced in glutamine deprivation condition in both tested cell types, moreover, this effect is amplified by the blockade of the ERN1 enzyme function. The expression levels of retinoblastoma, EID1, RBAP48, and E2F3 are decreased in glutamine deprivation condition only in ERN1-deficient glioma cells, but RBL1, CTIP, RBAP46, and E2F1-in both tested cell types with more significant effect in ERN1-deficient cells. Glucose deprivation condition leads to a decrease of expression levels of retinoblastoma, RBL1, E2F3, RBAP46, and RBAP48 in both used cell types and of EID1 and E2F1 only in glioma cells with suppressed function of signaling enzyme ERN1. Thus, expression levels of retinoblastoma and most retinoblastoma-related genes are increased under a blockade of ERN1 enzyme function and significantly changed in hypoxia, glucose or glutamine deprivation conditions both in control U87 cells and ERN1-deficient cells, but inhibition of the unfolded protein response sensor ERN1 predominantly enhances these effects. Moreover, it is possible that the induction of the expression of retinoblastoma and most retinoblastomarelated genes after knockdown of ERN1 plays an important role in suppression of glioma proliferation.

Effect of hypoxia on the expression of genes encoding insulin-like growth factors and some related proteins in U87 glioma cells without IRE1 function

“…Th e expression levels of insulin-like growth factors (IGF1 and IGF2), IGF1 receptor (IGF1R), IGF binding protein-4 (IGFBP4), and stanniocalcin 2 (STC2) mRNAs as well as ACTB mRNA were measured in control U87 glioma cells and cells with a defi ciency of IRE1 by quantitative polymerase chain reaction in real-time using qPCR "RotorGene RG-3000" (Corbett Research, Germany) and SYBRGreen Mix (ABgene, Th ermo Fisher Scientifi c, Epsom, Surrey, UK). QuaniTect Reverse Transcription Kit (QIA-GEN, Hilden, Germany) was used for cDNA synthesis as described previously (Minchenko et al 2012). Polymerase chain reaction was performed in triplicate.…”

Endoplasmic reticulum stress and angiogenesis

“…Стрес ендоплазматичного ретикулума та ангіогенез Це було чітко продемонстровано переважно на клітинах гліоми, найбільш агресивних із відомих злоякісних пухлин з вираженим ангіогенезом та посиленою інвазією клітин у нормальну паренхіму головного мозку [54,55,59,62]. Гіпоксія та сигнальний шлях стресу ЕР тісно пов'язані з процесами ангіогенезу та росту злоякісних пухлин.…”

“…Так, TIMPs інгібують металопротеїнази матриксу, які розщеплюють CTGF (фактор росту сполучної тканини, який ще має назву IGFBP8, протеїн 8, що зв'язується з подібним до інсуліну фактором росту) і таким чином реактивують VEGF [28]. Виключення функції ERN1, основного сенсорно-сигнального ензиму стресу ЕР, пригнічує ріст пухлин і змінює характер гіпоксичної регуляції експресії генів, що контролюють процеси гліколізу, ангіогенезу та проліферації [31,32,40,41,53,[55][56][57][58][59]. Залежність гіпоксичної регуляції експресії генів від функції сенсорно-сигнального ензиму ERN1 можна пояснити тим, що ефекти гіпоксії здебільшого опосередковані транскрипційним фактором HIF, а його вміст залежить не лише від вмісту кисню, а і від сигнальних шляхів, що опосередковують процеси проліферації, зокрема від PI3K, AKT та MAPK шляхів, які в свою чергу залежать від ERN1 сигнального шляху [3].…”

The vascular endothelial growth factor genes expression in glioma U87 cells is dependent from ERN1 signaling enzyme function

“…Total RNA was extracted from different tumor tissues and normal tissue counterparts as described [34]. RNA pellets were washed with 75% ethanol and dissolved in nuclease-free water.…”

“…QuaniTect Reverse Transcription Kit (QIAGEN, Germany) was used for cDNA synthesis as described previously [34]. Polymerase chain reaction was performed in triplicate.…”