Minchenko Oleksandr H, Hrebennykova Vita O, Viletska Yuliia M, Hnatiuk Oksana S, Sliusar Myroslava Y, Kozynkevych Halyna E, Minchenko Dmytro O
1Department of Molecular Biology, Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv, Ukraine.
2Department of Pediatrics, National Bohomolets Medical University, Kyiv, Ukraine.
Endocr Regul. 2025 Mar 12;59(1):1-9. doi: 10.2478/enr-2025-0001. Print 2025 Jan 1.
For the effective growth of malignant tumors, including glioblastoma, the necessary factors involve endoplasmic reticulum (ER) stress, hypoxia, and the availability of nutrients, particularly glucose. The ER degradation enhancing alpha-mannosidase like protein 1 (EDEM1) is involved in ER-associated degradation (ERAD) targeting misfolded glycoproteins for degradation in an N-glycan-independent manner. EDEM1 was also identified as a new modulator of insulin synthesis and secretion. The present study aims to investigate the regulation of the gene expression in U87MG glioblastoma cells by hypoxia and glucose or glutamine deprivations depending on the knockdown of ERN1 (endoplasmic reticulum to nucleus signaling 1) with the intent to reveal the role of ERN1 signaling in the regulation of this gene expression and function in tumorigenesis. The U87MG glioblastoma cells (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 (4 h). For glucose and glutamine deprivations, the cells were exposed to DMEM medium without glucose and glutamine, respectively, for 16 h. The expression level of the gene was studied by quantitative RT-PCR and normalized to the ACTB mRNA. It was found that inhibition of endoribonuclease and protein kinase activities of ERN1 led to down-regulation of gene expression in glioblastoma cells. Moreover, the expression of this gene was also decreased after silencing ERN1 in glioblastoma cells. At the same time, the expression of gene did not significantly change in cells with inhibited ERN1 endoribonuclease only. The expression of the gene was increased under hypoxia in control U87MG cells, but resistant to hypoxia in cells with ERN1 knockdown. Furthermore, the expression of this gene was up-regulated under glucose and glutamine deprivations in control glioblastoma cells. However, the ERN1 knockdown increased the sensitivity of gene expression to glucose and decreased to glutamine deprivations. The results of the present study demonstrate that inhibition of ERN1 down-regulated the expression of the gene through protein kinase activity of ERN1 and that the regulation of this gene expression by hypoxia and nutrient supply, especially glucose, is differently controlled by ERN1 in glioblastoma cells.
对于包括胶质母细胞瘤在内的恶性肿瘤的有效生长，必要因素包括内质网（ER）应激、缺氧以及营养物质尤其是葡萄糖的可利用性。内质网降解增强α-甘露糖苷酶样蛋白1（EDEM1）参与内质网相关降解（ERAD），以内切核糖核酸酶和蛋白激酶（dnERN1）或仅内质网内切核糖核酸酶（dnrERN1）。通过二甲基草酰甘氨酸（4小时）诱导缺氧。对于葡萄糖和谷氨酰胺剥夺，将细胞分别暴露于不含葡萄糖和谷氨酰胺的DMEM培养基中16小时。通过定量RT-PCR研究该基因的表达水平，并将其标准化为ACTB mRNA。结果发现，抑制ERN1的内切核糖核酸酶和蛋白激酶活性导致胶质母细胞瘤细胞中该基因表达下调。此外，在胶质母细胞瘤细胞中沉默ERN1后，该基因的表达也降低。同时，仅抑制ERN1内切核糖核酸酶的细胞中该基因的表达没有显著变化。在对照U87MG细胞中，缺氧时该基因的表达增加，但在ERN1敲低的细胞中对缺氧具有抗性。此外，在对照胶质母细胞瘤细胞中，葡萄糖和谷氨酰胺剥夺时该基因的表达上调。然而，ERN1敲低增加了该基因表达对葡萄糖的敏感性，并降低了对谷氨酰胺剥夺的敏感性。本研究结果表明，抑制ERN1通过ERN1的蛋白激酶活性下调该基因的表达，并且在胶质母细胞瘤细胞中，缺氧和营养供应尤其是葡萄糖对该基因表达的调节受ERN1的不同控制。
