High MIG-6 expression promotes tumor proliferation and metastasis of gastric cancer

Abstract

Background: Mitogen-inducible gene-6 (MIG-6) is a feedback inhibitor that targets activated epidermal growth factor receptor (EGFR) and suppresses tumor growth fueled by constitutively activated EGFR. Nevertheless, the action mechanism of MIG-6 in gastric cancer (GC) remains to be elucidated. Methods: Western blotting, fluorescence quantitative PCR, and immunohistochemistry were performed to detect the expression of MIG-6 in GC cell lines and tissues. Public databases were used to analyze MIG-6 in patients with GC. Furthermore, the GC cell lines were selected for the knockdown and overexpression of MIG-6. Results: Bioinformatics and histological analyses showed that MIG-6 was elevated in human GC tissues and cells. The Kaplan-Meier plotter showed that patients with elevated MIG-6 expression had significantly shorter survival. Furthermore, small interference RNA-mediated reduction of MIG-6 expression decreased EGFR/AKT signaling, as well as the proliferation and metastasis of human GC cells in vitro, whereas its overexpression increased these actions. Also, MIG-6 overexpression promoted the epithelial-mesenchymal transition of GC cells as well as the expression of the migration-associated protein matrix metalloproteinase-9 in vitro. Conclusion: These results suggest that MIG-6 can serve as a new prognostic biomarker or potential therapeutic target for the identification of patients with poor survival.

Keywords: EGFR; Gastric cancer; MIG-6; metastasis; proliferation; survival..

Figure 1

The expression of MIG-6 in the GC tissues and cell lines. (A) The mRNA level of MIG-6 in 43 pairs of GC tissues and paracancerous tissues was detected by qRT-PCR. (B) The mRNA level of MIG-6 in the paracancerous tissues (n = 38) and GC tissues (n = 46) from the ONCOMINE database. (C) Immunohistochemistry detected the protein level of MIG-6 in the paracancerous tissues (left panel) and GC tissues (right panel) (Original magnification: ×100). (D) The effect of MIG-6 mRNA expression on the overall survival and progression-free survival in GC patients was analyzed by Kaplan-Meier Plotter (http://www.kmplot.com). (E) and (F) The expression of MIG-6 in human gastric epithelial cells GES-1 and GC cell lines were detected by western blotting. *P < 0.05, **P < 0.01

Figure 2

The expression of MIG-6 was enhanced in BGC-823 cells and knocked down in SGC-7901 cells. (A) and (B) The expression of MIG-6 in SGC-7901 cells transfected with si-MIG-6 decreased significantly. **P < 0.01 versus SGC-7901 cells transfected with NC SGC-7901 cells. (C) and (D) pcDNA3.0-MIG-6 could efficiently increase the expression of MIG-6 in BGC-823 cells. **P < 0.01 versus blank BGC-823 cells.

Figure 3

The regulation of MIG-6 in the EGFR/AKT pathway. (A) and (B) The knockdown of MIG-6 inhibits EGFR and AKT phosphorylation expression in SGC-7901 cells. (C) and (D) The overexpression of MIG-6 promotes EGFR and AKT phosphorylation expression in BGC-823 cells, *P < 0.05, **P < 0.01.

Figure 4

The knockdown of MIG-6 reduced the ability of proliferation of SGC-7901 cells (A) and (B) Colony formation assay was used to detect the ability of proliferation in SGC-7901 cells after MIG-6 knockdown. (C) and (D) Colony formation assay was performed to detect the ability of proliferation in BGC-823 cells with MIG-6 overexpression. (E) and (F) CCK-8 assay was performed in BGC-823 and SGC-7901 cells to examine the cell proliferation ability (Scale bar = 100 μm), *P < 0.05, **P < 0.01.

Figure 5

The expression of MIG-6 affected EMT-associated proteins in BGC-823 and SGC-7901 cells. (A) and (B) Western blotting revealed EMT phenotype with reduced Snail, Vimentin, and N-cadherin and boosted the E-cadherin expression in SGC-7901 cells transfected with si-MIG-6. (C) and (D) EMT phenotype displayed that high MIG-6 expression could induce Snail, Vimentin, and N-cadherin expression and reduce the E-cadherin expression. (E) and (F) The expression of MMP9 was decreased by western blotting in SGC-7901 cells transfected with si-MIG-6. (G) and (H) The expression of MMP9 was increased by western blotting in BGC-823 cells transfected with pcDNA3.0-MIG-6. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

The migratory ability was evaluated by using transwell migration assay in BGC-823 and SGC-7901 cells. (A) and (B) The knockdown of MIG-6 reduced the migratory ability of SGC-7901 cells. (C) and (D) The overexpression of MIG-6 enhanced the migratory ability of BGC-823 cells. (200× magnification) **P < 0.01.