C-terminal of E1A binding protein 1 enhances the migration of gastric epithelial cells and has a clinicopathologic significance in human gastric carcinoma

Abstract

Background: Recent studies have claimed that the C-terminal of E1A binding proteins (CtBPs) influence tumorigenesis through participating in cell signal transduction in various human tumors. However, the detailed expression profiles of CtBP isoforms in human gastric cancer (GC) and the molecular mechanisms of CtBP involvement in tumor cell phenotypes warrant further investigation.

Materials and methods: The expression of CtBPs in GC cell lines and a human gastric epithelial cell line were explored via RT-qPCR and Western blotting assays. Moreover, the expression profiles of CtBPs in GC and histologically noncancerous tissues were explored by immunohistochemistry. To explore the effects of CtBP1 on the metastatic phenotype in GC, gastric epithelial cells were transfected with a eukaryotic expression plasmid to overexpress CTBP1, and the endogenous CtBP1 or JAK1 in GC cells was silenced through an RNA interference (RNAi) method. These transfections were validated via Western blotting, and the activation state of the JAK1/Stat3 signaling pathway was also explored via Western blotting. Furthermore, the malignant phenotype of GC cells was evaluated via a Cell Counting Kit-8 (CCK8) assay, colony formation assay, transwell assay, and wound-healing experiment.

Results: Our data revealed that the expression of CtBP1, but not CTBP2, was upregulated in 102 GC tissue samples compared with 98 noncancerous tissue samples, and the elevated expression level of CtBP1 was notably associated with distant metastasis. CTBP1 modulated cell migration and invasion through the JAK1/Stat3 signaling pathway in gastric epithelial cells. In addition, genetic silence of CtBP1 expression in GC cells notably constrained cell proliferation, invasion and migration abilities through inhibiting the activation of the JAK1/Stat3 pathway in GC cells.

Conclusion: Our data reveal that the knockout of CtBP1 notably constrains distant metastasis in GC through the JAK1/Stat3 pathway, suggesting that targeting CtBP1 is a practical anti-tumor approach to restrain tumor progression in GC.

Keywords: C-terminus of the E1A binding proteins; Janus Kinase 1; gastric cancer; signal transducer and activator of transcription 3.

Figure 1

The mRNA and protein expression levels of CTBP1 in human gastric epithelial cell line and GC cell lines. (A) The relative mRNA expression of CTBP1 in gastric epithelial cell line and GC cell lines. (B) The relative protein expression of CTBP1 in gastric epithelial cell line and GC cell lines. (C) The corresponding statistical analysis of CTBP1 protein expression. **p<0.01, compared with gastric epithelial cell line.

Figure 2

The expression of CTBP1 in GC patients. (A) Detection of CTBP1 in primary GC tissues and non-neoplastic tissues. (B) Protein expression of CTBP1 in human primary GC tissues and non-neoplastic tissues. (C) Kaplan-Meier Survival Curves and the Log-Rank Test were used in analysis of association between CTBP1 and survival time. (D) The corresponding statistical analysis of CTBP1 protein expression.

Figure 3

The overexpression of CtBP1 significantly promoted the malignant phenotype of gastric epithelial cell line cell. (A) Western blotting was utilized to examine the expression of CTBP1 and the activities level of the JAK1/Stat3 signaling pathway (left), and the corresponding statistical analysis of protein expression (right). Note: normalized with β-actin. (B) Growth curve of GES-1 cells detected by the CCK-8 assay. (C) The abilities of GES-1 cells to form colonies under 2D culture condition were determined through colony formation assay. (D) The Tanswell chambers method was utilized to explore the impact of CTBP1 overexpression on the invasive ability of GES-1 cells in vitro (left), and the corresponding statistical analysis (right). (E) The wound-healing assay was utilized to explore the migration ability of GES-1 cells in vitro. **p<0.01, compared with the vector group.

Figure 4

Loss of CtBP1 or JAK1 decreased the on the migration and invasion ability of GC cells. (A) Western blotting was used to examine the effects of silencing CtBP1 or JAK1 and the activation of the Stat3 signaling pathway in the SGC-790 cell line (left), and the corresponding statistical analysis of protein expression (right). Note: normalized with β-actin. (B) Growth curve of SGC-790 cells detected by the CCK-8 assay. (C)The abilities of SGC-790 cells to form colonies under 2D culture condition were determined through colony formation assay. (D) The Tanswell chambers method was utilized to explore the impact of CTBP1 or JAK1 slience on the invasive ability of SGC-790 cells in vitro (left), and the corresponding statistical analysis (right). (E) The wound-healing assay was utilized to explore the migration ability of SGC-790 cells in vitro. **p<0.01, compared with the scramble group.