Abstract
BackgroundIn cancer cells, transcriptional gene silencing has been associated with genetic and epigenetic defects. The disruption of DNA methylation patterns and covalent histone marks has been associated with cancer development. Until recently, microRNA (miRNA) gene silencing was not well understood. In particular, miR-125b1 has been suggested to be an miRNA with tumor suppressor activity, and it has been shown to be deregulated in various human cancers. In the present study, we evaluated the DNA methylation at the CpG island proximal to the transcription start site of miR-125b1 in cancer cell lines as well as in normal tissues and gynecological tumor samples. In addition, we analyzed the association of CTCF and covalent histone modifications at the miR-125b1 locus.MethodsTo assess the DNA methylation status of the miR-125b1, genomic DNA was transformed with sodium bisulfite, and then PCR-amplified with modified primers and sequenced. The miR-125b1 gene expression was analyzed by qRT-PCR using U6 as a control for constitutive gene expression. CTCF repressive histone marks abundance was evaluated by chromatin immunoprecipitation assays.ResultsThe disruption of CTCF in breast cancer cells correlated with the incorporation of repressive histone marks such H3K9me3 and H3K27me3 as well as with aberrant DNA methylation patterns. To determine the effect of DNA methylation at the CpG island of miR-125b1 on the expression of this gene, we performed a qRT-PCR assay. We observed a significant reduction on the expression of miR-125b1 in cancer cells in comparison with controls, suggesting that DNA methylation at the CpG island might reduce miR-125b1 expression. These effects were observed in other gynecological cancers, including ovarian and cervical tumors.ConclusionsA reduction of miR-125b1 expression in cancers, correlated with methylation, repressive histone marks and loss of CTCF binding at the promoter region.
Highlights
In cancer cells, transcriptional gene silencing has been associated with genetic and epigenetic defects
DNA methylation profile of the miR-125b1 CpG island in breast cancer cell lines As a first approach, we conducted an in silico study of the locus of miR-125b1
Using synthetic oligonucleotides modified for DNA methylation analysis, we determined that the CpG island associated with miR-125b1 is methylated in most breast tumor cell lines, with the exception of SK-BR-3 (Figure 1B, C)
Summary
Transcriptional gene silencing has been associated with genetic and epigenetic defects. The disruption of DNA methylation patterns and covalent histone marks has been associated with cancer development. MicroRNA (miRNA) gene silencing was not well understood. MiR-125b1 has been suggested to be an miRNA with tumor suppressor activity, and it has been shown to be deregulated in various human cancers. We evaluated the DNA methylation at the CpG island proximal to the transcription start site of miR-125b1 in cancer cell lines as well as in normal tissues and gynecological tumor samples. A hypermethylation of a CpG island located in proximity to the transcription initiation site was observed in cell lines and in tissue samples from patients with breast cancer [8,11]
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