[Promoter methylation and mRNA expression of WT1 gene in MCF10 breast cancer model]

To investigate the role of WT1 gene in breast carcinogenesis by analyses of the promoter methylation status and mRNA expression of WT1 gene in MCF10 model system of breast cancer progression.

Methylation specific PCR and sodium bisufite genomic sequencing were employed to detect methylation status of WT1 promoter in normal breast tissue, traditional breast cancer cell line MCF7 and MCF10 model series, including MCF10A (breast hyperplastic cell line, non-tumorigenic), MCF10AT (pre-malignant cell line, forming slowly progressing hyper and dysplastic lesions), MCF10DCIS.com (breast ductal carcinoma in situ cell line, forming ductal carcinoma in situ), and three invasive cell lines with metastatic potential (MCF10CA1a, MCF10CA1d, and MCF10CA1h). Real time reverse transcription PCR assay was used to determine the mRNA expression levels of WT1 in various cell lines.

Hypermethylation of WT1 promoter was identified in MCF7 and all MCF10 model cell lines (MCF10A, MCF10AT, MCF10DCIS.com, MCF10CA1a, MCF10CA1d, and MCF10CA1h). Unexpectedly, an increased expression of WT1 mRNA was found in all MCF10 cell lines and MCF7 comparing with normal breast tissue [folds of overexpression: 3.23 (MCF10A), 1.94 (MCF10AT), 4.20 (MCF10CA1a), 1.53 (MCF10CA1d), 4.20 (MCF10CA1h), 4.35 (MCF10DCIS) and 28.69 (MCF7)].

Promoter methylation does not silence the mRNA expression of WT1 during the development of breast cancer. Overexpression of WT1 occurs in the early stages of breast cancer development, suggesting its role as an oncogene rather than a tumor suppressor gene.