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

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.