High expression of eIF4A1 promotes angiogenesis through the NF‑κB/VEGFA pathway and predicts poor prognosis in gastric cancer

Abstract

Increased eukaryotic translation initiation factor 4A (eIF4A1) expression is observed in numerous types of cancer and is associated with carcinogenesis; however, little is known about the role of eIF4A1 in gastric cancer (GC) angiogenesis. In the present study, a total of 1,758 gastric mucosa samples were collected for immunohistochemical staining in tissue microarrays. The expression levels of eIF4A1 and their association with clinicopathological characteristics and prognosis were analyzed using χ² test and univariate/multivariate analysis. The effects of abnormal eIF4A1 expression in GC cells on proangiogenic activity was detected using the Cell Counting Kit‑8 proliferation assay, wound healing assay, Transwell assay, angiogenesis assay and a subcutaneous tumor model. The role of eIF4A1 in tumor‑infiltrating lymphocytes was explored using bioinformatics analysis. Furthermore, the effect of eIF4A1 on proangiogenic factors was confirmed by the quantitative polymerase chain reaction and western blotting. Notably, eIF4A1 was highly expressed in GC tissues, and was associated with patient age, tumor differentiation, depth of invasion, distant metastasis and Tumor‑Node‑Metastasis stage. Furthermore, it was suggested that high eIF4A1 expression could be regarded as a poor prognostic biomarker for patients with GC. The expression levels of eIF4A1 in GC cells were also positively related to proliferation, migration and the tube formation of human umbilical vein endothelial cells, and microvessel density in vivo. Furthermore, eIF4A1 in GC cells regulated the infiltration of immune cells in the tumor microenvironment, and promoted the expression of VEGFA and NF‑κB. In conclusion, eIF4A1 may promote GC angiogenesis through the NF‑κB/VEGFA pathway, and could be considered an independent prognostic biomarker for patients with GC.

Keywords: NF‑κB; VEGFA; angiogenesis; eukaryotic translation initiation factor 4A; gastric cancer; prognosis.

Figure 1.

eIF4A1 is upregulated in gastric cancer tissues. (A) Pan-cancer analysis of eIF4A1 expression was performed using UALCAN. (B) Expression of eIF4A1 in gastric cancer tissues and in peripheral normal tissues from patients with gastric cancer was assessed using Home For Researchers database. (C) Expression of eIF4A1 in late-stage GC tissues, early-stage GC tissues and in peripheral normal tissues. (D) Statistics of eIF4A1 expression in different types of gastric tissues. (E) eIF4A1 protein in GC tissues and normal gastric tissues: (a) Well-differentiated adenocarcinoma, (b) papillary adenocarcinoma, (c) poorly-differentiated adenocarcinoma, (d) mucinous adenocarcinoma, (e) CG, (f) pericarcinomatous mucosa tissue, (g) IM and (h) HGIN. *P<0.05, ***P<0.001, ****P<0.0001. CG, chronic gastritis; eIF4A1, eukaryotic translation initiation factor 4A; HGIN, high-grade intraepithelial neoplasia; IM, intestinal metaplasia; LGIN, low-grade intraepithelial neoplasia.

Figure 2.

Survival curves confirming the prognostic value of eIF4A1 and TNM stage in GC. (A) High eIF4A1 expression was associated with overall survival in patients with GC. (B) TNM stage was associated with the overall survival of patients with GC. GC, gastric cancer; TNM, Tumor-Node-Metastasis.

Figure 3.

Dysregulation of eIF4A1 affects the angiogenic activity of gastric cancer cells. (A) Western blotting detected the expression of eIF4A1 in cells after lentivirus infection. (B) Cell Counting Kit 8 assay assessed the proliferation of HUVECs treated with CM derived from specific cells. The optical density at 0 h was set at 100%. (C) Representative images (left) and quantification (right) of wound healing assay of HUVECs treated with CM derived from specific cells. (D) Representative images (left) and quantification (right) of Transwell assay of HUVECs treated with CM derived from specific cells. (E) Representative images (left) and quantification (right) of angiogenesis assay of HUVECs treated with CM derived from specific cells. (F) Representative image of the tumors. (G) Growth curves of the tumors and (H) weight of the tumors. (I) Representative images (left) and quantification (right) of CD31 staining in tissues from a subcutaneous xenograft tumor model. ^#P<0.05 vs. NC; ^&P<0.05 vs. shNC; *P<0.05. CM, conditioned media; eIF4A1, eukaryotic translation initiation factor 4A; HUVECs, human umbilical cord endothelial cells; MVD, microvessel density; NC, negative control; sh, short hairpin.

Figure 4.

eIF4A1 affects regulation of the TME and angiogenesis. Association between eIF4A1 expression and various component cells in the TME was detected using R package (4.3.3) in (A) GSE62254, (B) GSE15459 and (C) GSE84426. Part of the left panel was enlarged and presented in the right panel. (D) Correlations between eIF4A1 expression and CD31, VEGFA, NFKB1 and NFKB2. (E) mRNA levels of VEGF family genes were detected by reverse transcription-quantitative polymerase chain reaction. (F) Changes in VEGEF and NF-κB expression after eIF4A1 overexpression and knockdown were detected by western blotting. *P<0.05. eIF4A1, eukaryotic translation initiation factor 4A; NC, negative control; sh, short hairpin; TME, tumor microenvironment. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.