promoter hypermethylation and its effect on gene expression in human breast cancer.

Tumor-specific promoter hypermethylation of large tumor suppressor, homolog 2 (), a tumor suppressor gene, has been investigated using methylation-specific polymerase chain reaction (MSP) assays in different types of human cancer producing conflicting results. The aim of the present study was to evaluate the methylation status of the promoter region using bisulfite sequencing with a next generation sequencer for breast cancer. In the 11 patients enrolled in the present study, the promoter methylation index (MI) was uniformly high in tumor and normal tissues of the breast (median, 84.0 and 87.4%, respectively). The presence of promoter hypermethylation was confirmed in isolated tumor cells and normal epithelial cells using the magnetic-activated cell sorting method. hybridization for (mRNA) revealed that the mRNA expression of was higher in normal epithelial cells, compared with tumor cells, however, it was not significantly associated with MI. In 12 breast cancer cell (BCC) lines and two normal breast cell lines, the promoter was uniformly hypermethylated with no correlation between the mRNA expression of and the MI. In addition, treatment of the BCC lines with a demethylating reagent had minimal effect on the mRNA expression of in any of these cell lines. These results demonstrated that hypermethylation was not involved in silencing the mRNA expression of mRNA. The lower mRNA expression level of in tumor cells, compared with normal epithelial cells, suggested the possible involvement of downregulation in the mRNA expression of in the pathogenesis of breast cancer. Therefore, the conflicting results previously reported for promoter methylation in different types of cancer, detected using MSP assays may be attributable to the low fidelity of the MSP assay.