Figure 3.

Demethylation of the Cosmc promoter inhibits the expression of the Tn and STn antigens through the upregulation of Cosmc. (A) CpG island predicted by the MethPrimer website. MDA-MB-231 and MCF-7 cells were treated with methyltransferase M.SssI or methyltransferase inhibitor 5-aza-dc, with DMSO as control. (B) methylation of the Cosmc promoter region in MDA-MB-231 and MCF-7 cells detected by MSP. (C) methylation of Cosmc promoter region in MDA-MB-231 and MCF-7 cells determined by BSP. (D) enrichment of Dnmt1, Dnmt3a, and Dnmt3b in the Cosmc promoter region of MDA-MB-231 and MCF-7 cells evaluated by ChIP. (E and F) protein bands and expression of Cosmc, Tn and STn antigens in MDA-MB-231 and MCF-7 cells assessed by Western blot analysis. *p<0.05 vs normal breast epithelial cell line; ^#p<0.05 vs MDA-MB-231 and MCF-7 cells treated with DMSO. The above were measurement data and were expressed as mean ± standard deviation. Comparisons between two groups were performed using an independent sample t-test. All experiments were repeated 3 times.

Abbreviations: STn, Sialyl-Tn; DMSO, dimethyl sulfoxide; MSP, methylation specific polymerase chain reaction; ChIP, chromatin immunoprecipitation.