Disruption of CTCF at the miR-125b1 locus in gynecological cancers - PubMed (original) (raw)
Disruption of CTCF at the miR-125b1 locus in gynecological cancers
Ernesto Soto-Reyes et al. BMC Cancer. 2012.
Abstract
Background: In 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.
Methods: To 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.
Results: The 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.
Conclusions: A reduction of miR-125b1 expression in cancers, correlated with methylation, repressive histone marks and loss of CTCF binding at the promoter region.
Figures
Figure 1
Correlation between DNA methylation and expression status of miR-125b1 in breast cancer cell lines. A- Schematic representation of the locus of miR-125b1. Circles represent the CTCF binding sites, open circles represent the CpG island (CGI) present upstream of miR-125b1 and arrows represent the transcription start site. The regions amplified by PCR for the chromatin immunoprecipitation (ChIP) assay are represented in arrows and named by regions as RI to RIV. B- Assessment of DNA methylation status using MS-PCR of miRNAs in the breast cancer cell lines SK-BR-3, MDA-MB-231 and MCF7. As a control for the technique, we assessed the methylation state of DNA from non-tumorigenic epithelial cell line MCF 10A. DNA from lymphocytes was methylated in vitro by SssI methyltransferase, and used as a positive methylated DNA. M represents methylated, and U represents non-methylated. C- Determination of the DNA methylation status using sodium bisulfite and sequencing in breast cancer cell lines. Black boxes represent methylated CpGs, and white boxes represent non-methylated CpGs. D- Expression analysis of miR-125b1 in breast cancer cell lines compared with MCF 10A.P < 0.001 Student's _t_-test.
Figure 2
Analysis of the DNA methylation status in normal tissue and gynecological tumor samples. A- Analysis of DNA methylation by MS-PCR in several normal breast (NB), ovary (NO), cervix (NC) and peripheral blood lymphocytes (NL). Assessment of DNA methylation state in various gynecological primary tumors: Breast cancer (BC), ovarian cancer (OC) and cervical cancer (CC). B- Determination of the DNA methylation status using sodium bisulfite and sequencing in primary breast cancer samples and normal tissue samples. Black boxes represent methylated CpGs, and white boxes represent non-methylated CpGs. C- Expression analysis of miR-125b1 from primary breast cancer samples compared with normal breast tissue obtained during an aesthetic mammoplasty. **P < 0.001 Student's _t_-test.
Figure 3
In vivo CTCF binding analysis in miR-125b1 locus and the characterization of covalent histone marks in both normal cells and in primary breast tumors. A- Chromatin immunoprecipitation (ChIP) of CTCF and the histone covalent marks analyzed at the human miR-125b1 locus in cells obtained from normal breast tissue (NB 1). B- Characterization of CTCF and histone covalent marks in two tumor samples tissue from different patients (BC8 and BC9), input amplification refers as the entire population of DNA. A non-specific IgG antibody was used as a control. C- Positive controls for the ChIP assay. For CTCF we performed a PCR of the p53 gene promoter region in normal breast (NB1) and two tumor samples (BC8 and BC9); for H3K27me3 we evaluated the abundance of the H3K27me3 histone mark at the Myelin transcription factor 1 distal promoter (MYT1) region in two tumor samples (BC8 and BC9).
Figure 4
Model of miR-125b1 gene silencing, in which the absence of the nuclear factor CTCF is associated with DNA methylation of the CpG island, and the enrichment of repressive histone marks. In normal breast, CTCF might prevent the recruitment of the epigenetic silencing components, such as DNA methylation and repressive histone marks, also favors an open chromatin conformation (green color represent an open chromatin configuration). Meanwhile in breast cancer the loss of CTCF is associated with the CpG island (CGI) methylation and the gain of the repressive histone marks such as H3K9me3 and H3K27me3.
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