Methylation and demethylation of SOX2 gene promoter in BGC-823 gastric cancer cells

Abstract

To study the significance and effect of methylation status of sex determining region Y-box 2 (SOX2) gene promoter, and to investigate the effect of demethylation on the cell proliferation and invasion in BGC-823 gastric cancer cells. Methylation-specific PCR (MSP) was used to assess the role of 5-aza-2'-deoxycytidine (5-Aza-CdR) in the methylation of SOX2 promoter in the BGC-823 cell lines treated with different concentration of 5-Aza-CdR. We mapped the expression of SOX2 in the BGC-823 cell lines by the quantitative real-time PCR (qPCR) and Western blotting before and after treatment of 5-Aza-CdR. The survival of BGC-823 cells were detected by MTT assay. The invasion and migration of BGC-823 cells were investigated by transwell methods, and the migration of BGC-823 cells was also assessed by the scratch assay exposed to 5-Aza-CdR or vehicle control. Model of transplanted tumor on nude mouse were used to study the anticancer effect of 5-Aza-CdR in vivo by qPCR, Western blotting and immunohistochemistry. The methyltransferase inhibitor 5-Aza-CdR restored the loss of SOX2 expression in BGC-823 cell lines in a dose-dependent manner. The mRNA and protein expression of SOX2 had significant difference between the gastric cancer tissues and normal gastric mucosa (mRNA levels: 22.80±0.36 vs 20.36±0.45, P<0.05; protein levels: 0.49±0.01 vs 0.91±0.28, P<0.05). It also had significant difference among the BGC-823 cell lines treated with 5-Aza-CdR of the different concentrations (0, 1 and 10 μmol/L) (mRNA levels: 22.99±0.42 vs 21.78±0.41 vs 20.51±0.47, P<0.05; protein levels: 0.65±0.19 vs 0.73±0.13 vs 0.83±0.14, P<0.05). Compared with the control group (5-Aza-CdR concentration of 0 μmol/L), the survival rates of BGC-823 cell lines were significantly decreased in treatment groups (5-Aza-CdR concentrations of 1, 10 and 20 μmol/L, all P<0.05). Restored expression of SOX2 in the BGC-823 cell lines inhibited cell proliferation, colony formation and cell migration (P<0.05). Model of transplanted tumor on nude mouse in the 5-Aza-CdR group eventually had the smaller tumor size, the lighter tumor weight and the longer survival time than these in the PBS group [(286.6±37.5) vs (540.7±42.6)mm(3,) P<0.05; (325.2±32.2) vs (694.7±36.1)mg, P<0.05; (22.5±1.0) vs (18.7±1.6) d, P<0.05]. Meanwhile, the 5-Aza-CdR group increased the SOX2 protein expression levels and immunohistochemistry scores (0.96±0.25 vs 0.73±0.15, P<0.05; 6.23±0.45 vs 3.76±0.43, P<0.05). The SOX2 gene promotor is in the status of methylation in the BGC-823 cell. The recovery expression of SOX2 inhibits cell proliferation, invasion and the development of transplanted tumor in nude mice through DNA methyltransferase inhibition. It could suggest a new approach for the treatment of gastric cancer.