Epigenetic inactivation of microRNA genes in mammary carcinoma

Objective: This study addresses the question whether microRNA genes are affected by aberrant hypermethylation and subsequent transcriptional repression in human breast cancer.

Methods: All known human microRNA genes were screened for the association with CpG islands using different algorithms. Methylation status of candidate genes was assessed in a panel of breast cancer cell lines, various normal tissue samples and primary breast cancer specimen using COBRA, bisulfite sequencing and pyrosequencing. Transcriptional silencing was measured by real-time RT-PCR. The effect of demethylation on microRNA gene expression was analysed in breast cancer cell lines after treatment with the DNMT inhibitor 5'-deoxy-2-azacytidine.

Results: Aberrant hypermethylation was shown for mir-9-1, mir-124a3, mir-148, mir-152, and mir-663 in 34-86% of cases in a series of 71 primary human breast cancer specimens. miRNA gene hypermethylation correlated strongly with methylation of known tumour suppressor genes (p = 0.003). After treatment of various breast cancer cell lines with the demethylating agent 5-aza-2'deoxycytidine, reduction of mir-9-1 gene methylation and concomitant reactivation of expression could be observed. For the mir-9-1 gene, which is already hypermethylated in preinvasive intraductal lesions, a good correlation between quantitative methylation level and reduction of expression could be demonstrated in a subset of primary human breast cancer specimen (r = 0.8).

Conclusions: In addition to deletion and mutation, microRNA genes are also affected by aberrant hypermethylation in human breast cancer. This epigenetic inactivation is frequent and occurs early during breast cancer progression.