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. 2013;8(1):e54398.
doi: 10.1371/journal.pone.0054398. Epub 2013 Jan 16.

miRNA gene promoters are frequent targets of aberrant DNA methylation in human breast cancer

Lukas Vrba  1 , José L Muñoz-Rodríguez, Martha R Stampfer, Bernard W Futscher
Affiliations

miRNA gene promoters are frequent targets of aberrant DNA methylation in human breast cancer

Lukas Vrba et al. PLoS One. 2013.

Abstract

miRNAs are important regulators of gene expression that are frequently deregulated in cancer, with aberrant DNA methylation being an epigenetic mechanism involved in this process. We previously identified miRNA promoter regions active in normal mammary cell types and here we analyzed which of these promoters are targets of aberrant DNA methylation in human breast cancer cell lines and breast tumor specimens. Using 5-methylcytosine immunoprecipitation coupled to miRNA tiling microarray hybridization, we performed comprehensive evaluation of DNA methylation of miRNA gene promoters in breast cancer. We found almost one third (55/167) of miRNA promoters were targets for aberrant methylation in breast cancer cell lines. Breast tumor specimens displayed DNA methylation of majority of these miRNA promoters, indicating that these changes in DNA methylation might be clinically relevant. Aberrantly methylated miRNA promoters were, similar to protein coding genes, enriched for promoters targeted by polycomb in normal cells. Detailed analysis of selected miRNA promoters revealed decreased expression of miRNA linked to increased promoter methylation for mir-31, mir-130a, let-7a-3/let-7b, mir-155, mir-137 and mir-34b/mir-34c genes. The proportion of miRNA promoters we found aberrantly methylated in breast cancer is several fold larger than that observed for protein coding genes, indicating an important role of DNA methylation in miRNA deregulation in cancer.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Microarray analysis of DNA methylation status of miRNA promoters.
A, multidimensional scaling of pairwise distances derived from DNA methylation level of all miRNA promoter regions from all samples analyzed by microarray. CCL – cancer cell lines. B, same as A, but for in vitro grown cells only. C, same as A, but for tissue samples only. D, the proportion of miRNA promoters with DMRs in cancer cell lines. Displayed are data for all miRNA promoters, non-CTS promoters and CTS promoters. Hypermethylated promoters are in red, hypomethylated promoters in green and unchanged promoters in grey. E, the proportion of CTS miRNA promoters with DMRs in cancer cell lines. Displayed are data for all CTS miRNA promoters and those specific for either HMEC or HMF.
Figure 2
Figure 2. MicroRNA promoters hypermethylated in cancer cell lines are hypermethylated also in tumor tissue samples.
A, Venn diagram showing the overlap between non CTS miRNA promoters hypermethylated in cancer cell lines (CCL) and those hypermethylated in tumor tissue (TT) samples. p-value of the significance of the overlap is shown at the top. B, Hypermethylation status for 42 non-CTS miRNA promoters significantly hypermethylated in cancer cell lines group. Shown are data for individual cancer cell lines and individual tumor tissue samples analyzed by microarray. miRNA genes are listed on the left, sample names are on the top. miRNA promoters that are significantly hypermethylated in tumor tissue samples as a group are marked by an asterisk on the right. Additional miRNA promoters that are significantly hypermethylated in at least one tumor specimen are marked by a circle sign on the right. Cancer cell lines and tumor tissue samples are separated by vertical grey line. The mir-31 promoter of MB231 sample is grey because this cell line has a homozygous deletion of this region. mir-106a∼mir-363 designates mir-106a/mir-18b/mir-20b/mir-19b-2/mir-92a-2/mir-363 cluster and mir-941-1∼3 designates mir-941-1/mir-941-2/mir-941-3 cluster.
Figure 3
Figure 3. MassARRAY analysis of DNA methylation status of twelve selected miRNA promoters.
MassARRAY amplicons 350–550 bp in length were targeted to the regions, where DMRs were present in most cancer samples accordinng to microarray analysis. The data are represented as heatmaps with low percentage of methylation in yellow and high percentage of methylation in blue. The data of each miRNA promoter is shown in two panels; one for pure in vitro grown cells and the other for heterogenous tissue samples. The sample groups are labeled at the top. The columns contain data for individual samples labeled at the bottom. The rows show data from individual CpG units for MassARRAY amplicons. The six miRNA promoters that were analyzed on an expanded tumor sample set are at the top part of the figure. The data for the mir-31 promoter of MB231 sample are missing because this cell line has a homozygous deletion of this region.
Figure 4
Figure 4. Overview of DNA hypermethylation status for 12 miRNA promoters analyzed by MassARRAY.
miRNA genes are listed on the left, sample names are on the top. miRNA promoters that have significant ≥20% increase in DNA methylation within the whole MassARRAY amplicon relative to normal samples are in black. For a select set of miRNAs, 14 additional breast cancer specimens were analyzed. Cancer cell lines and tumor tissue samples are separated by vertical grey line. The mir-31 promoter of MB231 sample is grey because this cell line has a homozygous deletion of this region.
Figure 5
Figure 5. MicroRNA promoters occupied by polycomb specific H3K27me3 in normal cells are frequently hypermethylated in cancer.
A, Venn diagram showing the overlap between promoters occupied by H3K27me3 in ESC and those hypermethylated in breast cancer cell lines (CCL). B, Venn diagram showing the overlap between promoters occupied by H3K27me3 in HMEC and those hypermethylated in breast CCL. Both overlaps are highly significant (hypergeometric test).
Figure 6
Figure 6. Aberrant DNA methylation of miRNA gene promoters in cancer is linked to silencing of miRNA expression.
Each plot displays the relation between DNA methylation differences and miRNA expression differences from control in individual samples including breast cancer cell lines, breast tumor specimens, three normal HMEC, and normal breast tissue samples. The x-axis shows the difference in DNA methylation (%) between the sample and a normal reference for the whole MassARRAY amplicon. The y-axis shows the difference in miRNA expression (dCt) between the sample and a normal reference as determined by real-time PCR analysis. The names of miRNA genes/clusters analyzed by MassARRAY and the names of respective mature miRNA products detected by real-time PCR are displayed at the top of each plot. The Spearman correlation coefficient rho and the p-value (two-sided) of the correlation are displayed at the second line at the top of each plot.
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