The impact of ERN1 endoribonuclease activity inhibition on TOB1, HBEGF, and TWIST1 genes expression in U87MG glioblastoma cells.

Q3 Medicine Endocrine regulations Pub Date : 2025-03-12 Print Date: 2025-01-01 DOI:10.2478/enr-2025-0004
Oleksandr H Minchenko, Myroslava Y Sliusar, Yuliia M Viletska, Olha V Rudnytska, Denys V Kolybo
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Abstract

Objective. It is known that inhibition of the endoplasmic reticulum transmembrane signaling protein (ERN1) suppresses the glioblastoma cells proliferation. The present study aims to investigate the impact of inhibition of ERN1 endoribonuclease and protein kinase activities on the TOB1, HBEGF, and TWIST1 gene expression in U87MG glioblastoma cells with an intent to reveal the role of ERN1 signaling in the regulation of expression of these genes. Methods. The U87MG glioblastoma cells with inhibited ERN1 endoribonuclease (dnrERN1) or both enzymatic activities of ERN1 (endoribonuclease and protein kinase; dnERN1) were used. Cells transfected with empty vector served as controls. Wild-type glioblastoma cells were used for mRNA silencing. The expression level of the TOB1, HBEGF, and TWIST1 genes and microRNA were studied by quantitative RT-PCR. Results. We found that inhibition of ERN1 endoribonuclease activity led to a strong down-regulation of HBEGF gene expression in glioblastoma cells and did not significantly change the expression of TOB1 and TWIST1 genes. At the same time, inhibition of both enzymatic activities of ERN1 strongly increased the expression of the TOB1 gene and down-regulated HBEGF and TWIST1 genes in glioblastoma cells. The expression of TWIST1 gene increased, but HBEGF and TOB1 genes significantly decreased in cells with silencing of ERN1 mRNA by specific siRNA. At the same time, silencing of XBP1 mRNA reduced the expression of HBEGF gene only. In addition, in glioblastoma cells with ERN1 knockdown, the level of miR-96-5p was suppressed, but miR-182-5p was increased and could promote post-transcriptional expression of TWIST1, HBEGF, and TOB1 mRNAs. Conclusion. The results of the present study demonstrate that inhibition of ERN1 strongly up-regulated the expression of the anti-proliferative TWIST1 gene through protein kinase activity of ERN1 and that decreased HBEGF and TOB1 genes expression was also controlled preferentially by ERN1 protein kinase activity. These changes in the expression level of TWIST1, HBEGF, and TOB1 genes may also contribute to ERN1 knockdown-mediated suppression of glioblastoma cells proliferation.

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研究目的众所周知,抑制内质网跨膜信号蛋白(ERN1)可抑制胶质母细胞瘤细胞的增殖。本研究旨在探讨抑制ERN1内切酶和蛋白激酶活性对U87MG胶质母细胞瘤细胞中TOB1、HBEGF和TWIST1基因表达的影响,以期揭示ERN1信号转导在这些基因表达调控中的作用。研究方法使用抑制了ERN1内切核酸酶(dnrERN1)或ERN1两种酶活性(内切核酸酶和蛋白激酶;dnERN1)的U87MG胶质母细胞瘤细胞。转染空载体的细胞作为对照组。野生型胶质母细胞瘤细胞用于 mRNA 沉默。通过定量 RT-PCR 研究了 TOB1、HBEGF 和 TWIST1 基因及 microRNA 的表达水平。结果发现我们发现,抑制 ERN1 内切核酸酶活性会导致胶质母细胞瘤细胞中 HBEGF 基因表达的强烈下调,而对 TOB1 和 TWIST1 基因的表达没有显著改变。同时,抑制 ERN1 的两种酶活性会强烈增加胶质母细胞瘤细胞中 TOB1 基因的表达,并下调 HBEGF 和 TWIST1 基因的表达。用特异性 siRNA 沉默 ERN1 mRNA 的细胞中,TWIST1 基因的表达增加,但 HBEGF 和 TOB1 基因的表达明显减少。同时,沉默 XBP1 mRNA 只降低了 HBEGF 基因的表达。此外,在敲除 ERN1 的胶质母细胞瘤细胞中,miR-96-5p 的水平被抑制,但 miR-182-5p 水平升高,并能促进 TWIST1、HBEGF 和 TOB1 mRNA 的转录后表达。结论本研究结果表明,抑制 ERN1 可通过 ERN1 蛋白激酶活性强烈上调抗增殖 TWIST1 基因的表达,而 HBEGF 和 TOB1 基因表达的降低也优先受 ERN1 蛋白激酶活性的控制。TWIST1、HBEGF和TOB1基因表达水平的这些变化也可能是ERN1敲除介导的胶质母细胞瘤细胞增殖抑制的原因。
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来源期刊
Endocrine regulations
Endocrine regulations Medicine-Endocrinology, Diabetes and Metabolism
CiteScore
2.70
自引率
0.00%
发文量
33
审稿时长
8 weeks
期刊最新文献
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