Bone morphogenetic protein and activin membrane-bound inhibitor overexpression inhibits gastric tumor cell invasion via the transforming growth factor-β/epithelial-mesenchymal transition signaling pathway

Abstract

Gastric carcinoma is one of the most common human malignancies and remains the second leading cause of cancer-associated mortality worldwide. Gastric carcinoma is characterized by early-stage metastasis and is typically diagnosed in the advanced stage. Previous results have indicated that bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) overexpression has been demonstrated to inhibit growth and metastasis of gastric cancer cells. However, the molecular mechanisms of the BAMBI-mediated signaling pathway in the progression of gastric cancer are poorly understood. In the present study, to assess whether BAMBI overexpression inhibited the growth and aggressiveness of gastric carcinoma cells through regulation of transforming growth factor (TGF)-β/epithelial-mesenchymal transition (EMT) signaling pathway, the growth and metastasis of gastric carcinoma cells were analyzed following BAMBI overexpression and knockdown in vitro and in vivo. Molecular changes in the TGF-β/EMT signaling pathway were studied in gastric carcinoma cells following BAMBI overexpression and knockdown. DNA methylation of the gene regions encoding the TGF-β/EMT signaling pathway was investigated in gastric carcinoma cells. Tumor growth in tumor-bearing mice was analyzed after mice were subjected to endogenous overexpression of BAMBI. Results indicated that BAMBI overexpression significantly inhibited gastric carcinoma cell growth and aggressiveness, whereas knockdown of BAMBI significantly promoted its growth and metastasis compared with the control (P<0.01). The TGF-β/EMT signaling pathway was downregulated in BAMBI-overexpressed gastric carcinoma cells; however, signaling was promoted following BAMBI knockdown. In addition, it was observed that BAMBI overexpression significantly downregulated the DNA methylation of the gene regions encoding the TGF-β/EMT signaling pathway (P<0.01). Furthermore, RNA interference-mediated BAMBI overexpression also promoted apoptosis in gastric cancer cells and significantly inhibited growth of gastric tumors in murine xenografts (P<0.01). In conclusion, the present findings suggest that BAMBI overexpression inhibited the TGF-β/EMT signaling pathway and suppressed the invasiveness of gastric tumors, suggesting BAMBI may be a potential target for the treatment of gastric carcinoma via regulation of the TGF-β/EMT signaling pathway.

Keywords: DNA methylation; bone morphogenetic protein and activin membrane-bound inhibitor; gastric carcinoma; transforming growth factor-β/epithelial-mesenchymal transition.

Figure 1.

Effect of BAMBI expression on growth and aggressiveness in gastric cancer cells. (A) Protein expression levels of BAMBI in gastric cancer and normal gastric cells was assessed. Protein expression levels of BAMBI following transfection of (B) pBAMBI or (C) Si-BAMBI were determined. Endogenous BAMBI overexpression inhibited BGC-823 (D) cell growth, (E) migration and (F) invasion. Magnification, ×40. One-way analysis of variance or two-tailed Student's t-test were performed. Control, pvector. **P<0.01 as indicated. BAMBI, bone morphogenetic protein and activin membrane-bound inhibitor; Si, small interfering RNA; pBAMBI, overexpressed BAMBI.

Figure 2.

BAMBI regulated the growth and aggressiveness of gastric cancer cells through the TGF-β/EMT signaling pathway. (A) Proliferation marker IPO-38 and Ki67 protein expression levels in BGC-823 cells following BAMBI overexpression were assessed. (B) Intracellular pH changes in BAMBI-overexpressed BGC-823 cells were analyzed. (C) EMT marker TWIST1, MMP9 and SOX4 protein expression levels in BGC-823 cells following transfection with BAMBI were indicated. (D) EMT transcription factor SNAI1, ACTA2 and VIM protein expression levels in BGC-823 cells following transfection with BAMBI were determined. Effects of BAMBI (E) overexpression or (F) knockdown on TGF-β and its regulatory molecules expression levels of N-cadherin, CT-I and FIB in BGC-823 cells. Effects of BAMBI (G) overexpression or (H) knockdown on TGF-β-induced EMT signal pathway in BGC-823 cells. One-way analysis of variance or two-tailed Student's t-test were performed. **P<0.01 as indicated. Control, pvector. BAMBI, bone morphogenetic protein and activin membrane-bound inhibitor; TGF-β, transforming growth factor-β; EMT, epithelial-mesenchymal transition; Si, small interfering RNA; pBAMBI, overexpressed BAMBI; TWIST1, twist-related protein 1; MMP9, matrix metallopeptidase; SOX4, SRY-box 4; ACTA2, α-actin-2; VIM, vimentin; FIB, fibronectin; CT-I; collagen-I.

Figure 3.

Analysis of the association between BAMBI and DNA methylation. (A) DNA methylation analysis of TGF-β signaling pathway genes in gastric cancer cells were assessed following transfection with BAMBI. (B) Expression levels of BAMBI mRNA negatively correlated with increased methylation in BGC-823 cells (change from grey-blue-yellow indicated methylation was increased). (C) Percent of epigenetic modifications of the CpG loci located between the control and BAMBI group. (D) Scatter plot of DNA methylation values determined by HumanMethylation450K BeadChip analysis and bisulfite pyrosequencing. Pearson correlation coefficient, r=0.926. Effects of (E) BAMBI overexpression or (F) knockdown on the protein expression levels of Smad6 and Smad7 in BGC-823 cells were determined using western blot analysis. Effects of (G) BAMBI overexpression or (H) knockdown on TGF-β-induced phosphorylation of MAPK and Smad2 in BGC-823 cells were also assessed. One-way analysis of variance or two-tailed Student's t-test revealed a significant effect. Control, pvector. **P<0.01 as indicated. BAMBI, bone morphogenetic protein and activin membrane-bound inhibitor; TGF-β, transforming growth factor-β; Si, small interfering RNA; pBAMBI, overexpressed BAMBI; MAPK, mitogen-activated protein kinase; p, phosphorylated; Smad, mothers against decapentaplegic homolog.

Figure 4.

In vivo effects of BAMBI overexpression on gastric tumor progression. (A) Overexpression of BAMBI inhibited gastric cancer tissues growth. (B) Endogenic BAMBI overexpression improved intracellular pH in gastric tumors from xenograft mice. (C) Protein expression levels of ki67 and HIF in BAMBI overexpressed tumors were assessed. (D) TWIST1, MMP9 and SOX4 protein expression levels involved in the EMT signaling pathway in tumors were overexpressed BAMBI. (E) Protein expression levels of N-cadherin, CT-I and FIB were also assessed in BAMBI-overexpressed gastric tumors. (F) Apoptosis rate in gastric tumors transfected with BAMBI in vivo were indicated. White arrows indicate the apoptotic cells. One-way analysis of variance or two-tailed Student's t-test revealed a significant effect. **P<0.01 as indicated. BAMBI, bone morphogenetic protein and activin membrane-bound inhibitor; TGF-β, transforming growth factor-β; Si, small interfering; pBAMBI, overexpressed BAMBI; TWIST1, twist-related protein 1; MMP9, matrix metallopeptidase; SOX4, SRY-box 4; ACTA2, α-actin-2; VIM, vimentin; FIB, fibronectin; CT-I; collagen-I; HIF, hypoxia-inducible factor.