Eukaryotic initiation factor 4 A-3 promotes glioblastoma growth and invasion through the Notch1-dependent pathway

Abstract

Background: As an adult tumor with the most invasion and the highest mortality rate, the inherent heterogeneity of glioblastoma (GBM) is the main factor that causes treatment failure. Therefore, it is important to have a deeper understanding of the pathology of GBM. Some studies have shown that Eukaryotic Initiation Factor 4A-3 (EIF4A3) can promote the growth of many people's tumors, and the role of specific molecules in GBM remains unclear.

Methods: The correlation between the expression of EIF4A3 gene and its prognosis was studied in 94 GBM patients using survival analysis. Further in vitro and in vivo experiments, the effect of EIF4A3 on GBM cells proliferation, migration, and the mechanism of EIF4A3 on GBM was explored. In addition, combined with bioinformatics analysis, we further confirmed that EIF4A3 contributes to the progress of GBM.

Results: The expression of EIF4A3 was upregulated in GBM tissues, and high expression of EIF4A3 is associated with poor prognosis in GBM. In vitro, knockdown of EIF4A3 significantly reduced the proliferation, migration, and invasion abilities of GBM cells, whereas overexpression of EIF4A3 led to the opposite effect. The analysis of differentially expressed genes related to EIF4A3 indicates that it is involved in many cancer-related pathways, such as Notch and JAK-STAT3 signal pathway. In Besides, we demonstrated the interaction between EIF4A3 and Notch1 by RNA immunoprecipitation. Finally, the biological function of EIF4A3-promoted GBM was confirmed in living organisms.

Conclusion: The results of this study suggest that EIF4A3 may be a potential prognostic factor, and Notch1 participates in the proliferation and metastasis of GBM cells mediated by EIF4A3.

Keywords: EIF4A3; Glioblastoma; Invasion; Migration; Notch; Prognosis; Proliferation.

Fig. 1

Upregulation of EIF4A3 expression levels suggests a poor prognosis in GBM. (a) EIF4A3 was up-regulated in GBM based on The Cancer Genome Atlas. (b) The receiver operating characteristic (ROC) curve of EIF4A3 as a biomarker of GBM in The Cancer Genome Atlas. (c) EIF4A3 expression in tumor sections of different GBM patients with immunoglobulin as control and magnification. (d) High expression of EIF4A3 mediates poor prognosis in GBM patients

Fig. 2

Effect of EIF4A3 knockdown on the proliferation of T98G and U87-MG cells. (a) The expression of EIF4A3 in U87-MG and T98G cells after EIF4A3 knockdown (EIF4A3-KD) was determined by Western blotting. β-actin was used as an experimental control. (b) The proliferation of EIF4A3-KD and NC-treated U87-MG and T98G cells. (c) Proliferation of T98G and U87-MG cell lines in EIF4A3-KD and NC as determined by 5-ethynyl-2′-deoxyuridine (EdU). Scale bar: 20 μm

Fig. 3

Effect of EIF4A3 knockdown or overexpression on the migratory capacity of GBM cells. (a) Migration ability of U87-MG and T98G cells treated with EIF4A3 knockdown (EIF4A3-KD) and negative control (NC). Scale bar: 20 μm. (b) EIF4A3 overexpression (EIF4A3-OE) and migration ability of NC-treated U251-MG and A172 cells. Scale bar: 20 μm

Fig. 4

Effect of high EIF4A3 expression on signal transducer and activator of STAT3-related pathways in GBM cells. (a) Quadrant plot. The genes in quadrant 1 were upregulated genes consistent with EIF4A3 expression. (b) Biological process (BP) enrichment analysis was carried out for the continuously up-regulated genes. (c) Analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was performed on the consistently up-regulated genes. (d) EIF4A3 regulates Notch1 and STAT3 biological pathways. (e) Protein expression levels of STAT3-related signaling pathway genes in A172 and U251-MG cells with EIF4A3 overexpression (EIF4A3-OE) and in U87-MG and T98G cells with EIF4A3 knockdown (EIF4A3-KD). (f) EIF4A3 may regulate Notch signaling pathway and JAK − STAT signaling pathway to promote the migration and invasion of GBM cells

Fig. 5

Notch1 is involved in EIF4A3-mediated GBM cell proliferation and migration. (a) The proliferation of U251-MG and A172 cells overexpressing EIF4A3 was analyzed in the presence of a NC and DAPT. (b) Similarly, the proliferation of the cells was verified, showing representative fluorescence micrographs. Scale bar: 20 μm. (c-d) Transwell analysis of blank control and DAPT. Scale bar: 20 μm

Fig. 6

Effect of reduced EIF4A3 on GBM progression in GBM xenografts. (a) Intracranial injection of lentivirus-transformed U87-MG cells with NC or EIF4A3-KD into nude mice and U87-MG-NC cells with DAPT added. Animals were examined by magnetic resonance imaging. (b) Representative micrographs of hematoxylin-eosin-stained tumor sections. (c-d) Immunohistochemistry of tumor tissues stained with anti-EIF4A3 and anti-Notch1 antibody. Scale bar: 20 μm