EGFLAM correlates with cell proliferation, migration, invasion and poor prognosis in glioblastoma

Abstract

EGFLAM as a novel gene biomarker has been reported in some cancers but not glioblastoma (GBM) yet. To clarify the functional role of EGFLAM in GBM, we performed this study. Firstly, based on TCGA and Oncomine database, EGFLAM expression and clinical significance in GBM patients was analyzed. Furthermore, the biological effect of EGFLAM in GBM cells was determined by qRT-PCR, CCK-8 assay, colony formation assay, wound healing assay, transwell assays and western blot analysis. The databases analysis showed that EGFLAM expression was at higher levels in GBM patients with poor prognosis. The results indicated that EGFLAM silence inhibited the proliferation, migration and invasion of U87 cells, which was regulated through repression of PI3K/AKT pathway. Accordingly, the data from our work shed some light on EGFLAM might be a prognostic biomarker and therapeutic target for GBM.

Keywords: AKT; EGFLAM; PI3K; glioblastoma; prognosis; proliferation.

Figure 1.

EGFLAM expression was up-regulated in Glioblastoma (GBM). (A) The Cancer Genome Atlas (TCGA) database was used to analyze the expression levels of EGFLAM in GBM tissues and normal brain tissues. (B) Oncomine database was used to further determine the expression levels of EGFLAM in GBM tissues and normal brain tissues. (C) Kaplan-Meier method and the long-rank test were used to analyze the overall survival of patients, and patients with high expression of EGFLAM presented the worse prognosis ($p=$ 0.005).

Figure 2.

EGFLAM expression was up-regulated in GBM cells. (A) The expression levels of EGFLAM in HEB, U87, U251, HS683 cell lines were analyzed by qRT-PCR, among which U87 cell line was chosen as appropriate cellular models to knockdown EGFLAM for further experiments. (B–D) After transfection, EGFLAM expression was measured by qRT-PCR (B) and western blot (C and D) respectively. si-EGFLAM group represents EGFLAM in U87 cells were silenced, and si-con group represents EGFLAM in U87 cells were not silenced. *$p<$ 0.05 and **$p<$ 0.01 versus si-con group.

Figure 3.

EGFLAM promoted the proliferation of U87 cells. (A) Knockdown of EGFLAM inhibited the viability of U87 cells, which was determined by CCK-8 assays. (B) Knockdown of EGFLAM decreased the colony numbers of U87 cells, which was measured by colony formation assays (Original magnification $\times$ 2). si-EGFLAM group represents EGFLAM in U87 cells were silenced, and si-con group represents EGFLAM in U87 cells were not silenced. **$p<$ 0.01 versus si-con group.

Figure 4.

EGFLAM promoted the migration and invasion of U87 cells. (A) Wound healing assay revealed that knockdown of EGFLAM inhibited the migration of U87 cells (Original magnification $\times$ 100). (B) Transwell assays showed that knockdown of EGFLAM significantly suppressed the migration and invasion of U87 cells (Original magnification $\times$ 200). si-EGFLAM group represents EGFLAM in U87 cells were silenced, and si-con group represents EGFLAM in U87 cells were not silenced. **$p<$ 0.01 versus si-con group.

Figure 5.

EGFLAM induced the activation of PI3K/AKT pathway. (A) The expression levels of PI3K, p-PI3K (Tyr199), AKT, p-AKT (Ser473) and p-P70S6K (Ser371) in PI3K/AKT pathway were measured by western blot. (B) The expression of proteins in PI3K/AKT pathway was quantified and normalized to the GAPDH as a loading control. si-EGFLAM group represents EGFLAM in U87 cells were silenced, and si-con group represents EGFLAM in U87 cells were not silenced. **$p<$ 0.01 versus si-con group.