Department of Molecular Biology, Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv, Ukraine.
Endocr Regul. 2024 Oct 1;58(1):206-214. doi: 10.2478/enr-2024-0024. Print 2024 Jan 1.
Carboxypeptidase E (CPE) plays an important role in the biosynthesis of neurotransmitters and peptide hormones including insulin. It also promotes cell proliferation, survival, and invasion of tumor cells. The endoplasmic reticulum stress, hypoxia, and nutrient supply are significant factors of malignant tumor growth including glioblastoma. There are data indicating that the knockdown of the endoplasmic reticulum to nucleus signaling 1 (ERN1) suppressed glioblastoma cell proliferation and increased invasiveness of these cells. The present study aims to investigate the regulation of the gene in U87MG glioblastoma cells by ERN1 knockdown, hypoxia, and glucose or glutamine deprivations with the intent to reveal the role of ERN1 signaling in the regulation of this gene expression and function in tumorigenesis. Human glioblastoma cells U87MG (transfected by an empty vector; control) and ERN1 knockdown cells with inhibited ERN1 endoribonuclease and protein kinase (dnERN1) or only ERN1 endoribonuclease (dnrERN1) were used. Hypoxia was introduced by dimethyloxalylglycine; for glucose and glutamine deprivations, the cells were cultured in DMEM medium without glucose or glutamine for 16 h, respectively. The expression level of the gene was studied by quantitative RT-PCR and normalized to ACTB. It was found that inhibition of endoribonuclease and protein kinase activities of ERN1 led to a strong up-regulation of gene expression in glioblastoma cells. The expression of this gene also increased in glioblastoma cells after silencing ERN1. At the same time, the expression of this gene did not significantly change in cells with inhibited ERN1 endoribonuclease only. The expression of the gene was resistant to hypoxia in control U87MG cells, but increased in cells with ERN1 knockdown. The expression of this gene was up-regulated under glutamine deprivation in control glioblastoma cells, but decreased upon ERN1 knockdown. However, glucose deprivation decreased the expression of gene in both types of used cells, but ERN1 inhibition enhanced this effect. The results of the present study demonstrate that inhibition of ERN1 strongly up-regulated the expression of pro-oncogenic gene through protein kinase activity of ERN1 and that increased gene expression possibly participates in ERN1 knockdown-mediated invasiveness of glioblastoma cells.
羧肽酶 E（CPE）在包括胰岛素在内的神经递质和肽激素的生物合成中发挥重要作用。它还促进肿瘤细胞的增殖、存活和侵袭。内质网应激、缺氧和营养供应是包括神经胶质瘤在内的恶性肿瘤生长的重要因素。有数据表明，敲低内质网到核信号 1（ERN1）抑制神经胶质瘤细胞增殖并增加这些细胞的侵袭性。本研究旨在探讨 ERN1 敲低、缺氧以及葡萄糖或谷氨酰胺剥夺对 U87MG 神经胶质瘤细胞中 基因的调控，旨在揭示 ERN1 信号在肿瘤发生中调节该基因表达和功能的作用。 使用转染空载体的人神经胶质瘤细胞 U87MG（对照）和 ERN1 敲低细胞（ERN1 内切核酸酶和蛋白激酶被抑制的 dnERN1）或仅 ERN1 内切核酸酶被抑制的 dnrERN1）。通过二甲草酰基甘氨酸引入缺氧；对于葡萄糖和谷氨酰胺剥夺，将细胞分别在不含葡萄糖或谷氨酰胺的 DMEM 培养基中培养 16 小时。通过定量 RT-PCR 研究 基因的表达水平，并标准化为 ACTB。 结果发现，ERN1 内切核酸酶和蛋白激酶活性的抑制导致神经胶质瘤细胞中 基因表达的强烈上调。沉默 ERN1 后，该基因在神经胶质瘤细胞中的表达也增加。同时，仅抑制 ERN1 内切核酸酶时，该基因的表达没有明显变化。在对照 U87MG 细胞中，该基因的表达对缺氧不敏感，但在 ERN1 敲低的细胞中增加。在对照神经胶质瘤细胞中，谷氨酰胺剥夺会使该基因的表达上调，但在 ERN1 敲低时会降低。然而，葡萄糖剥夺会降低两种类型使用的细胞中 基因的表达，但 ERN1 抑制增强了这种效应。 本研究的结果表明，通过 ERN1 的蛋白激酶活性强烈抑制 ERN1 可上调致癌基因 基因的表达，并且增加的 基因表达可能参与 ERN1 敲低介导的神经胶质瘤细胞侵袭。
