DNA methylation contributes to deregulation of 12 cancer-associated microRNAs and breast cancer progression

Highlights

• Hypermethylation of MIR-125b-1, -127, -132, -193a, and -34b was revealed in BC

• Hypomethylation of MIR-191 was shown in primary breast tumors

• MIR-124-1, 125b-1, 127, 132, 193a, 34b methylation correlated with down-regulation

• Levels of miR-127-5p, -124-3p, -375 and DAPK1, BCL2, RASSF1(A) correlated negatively

• Hypermethylation of MIR-127 and -125b-1 was associated with breast cancer progression

Abstract

The methylation of promoter CpG islands and the interaction between microRNAs (miRNAs) and messenger RNAs (mRNAs) of target genes are considered two crucial mechanisms for gene and pathway deregulation in malignant tumors. The aim of this study was to analyze the role of promoter methylation in altering the expression of 13 miRNAs that are associated with breast cancer (BC): miR-124, -125b, -127, -132, -137, -148a, -191, -193a, -203, -212, -34b, -375, -9. The role of methylation in the deregulation of these miRNAs has not been previously assessed in the representative set of BC samples. We used a set of 58 paired (tumor/normal) breast tissue samples and methylation-specific PCR to demonstrate significant aberrations in the methylation patterns of 9 miRNA genes. In particular, we observed hypermethylation of MIR-127, -132, and -193a, and hypomethylation of MIR-191 for the first time. Using quantitative PCR, we established a strong correlation between promoter methylation and expression levels for 12 miRNA genes (all except MIR-212); this finding demonstrates the functional importance of altered methylation patterns. We also performed a correlation analysis between expression levels of the 13 miRNAs and 5 cancer-associated genes, namely RASSF1(A), CHL1, APAF1, DAPK1, and BCL2, which were predicted as targets for these miRNAs, to investigate the impact of these miRNAs on these genes with key cellular functions in BC. Significant negative correlation was revealed for the following miRNA-mRNA pairs: miR-127-5p and DAPK1, miR-375 and RASSF1(A), and miR-124-3p and BCL2. Additionally, we also found a strong association between hypermethylation of MIR-127 and MIR-125b-1 and BC progression, particularly metastasis. Thus, our findings provide evidence for the significant role of methylation in the deregulation of 12 miRNA genes in BC, identify putative novel functional miRNA-mRNA pairs, and suggest MIR-127 and MIR-125b-1 hypermethylation to be potential biomarkers of BC metastasis.

Introduction

DNA methylation of regulatory sequences at the genomic level and interaction between microRNAs (miRNAs) and the messenger RNAs (mRNAs) of target genes at the posttranscriptional level contribute to the dynamic regulation of gene activity. Aberrations in these mechanisms can lead to impaired functioning of cell signaling pathways, such as that observed in malignant tumors (Lopez-Serra and Esteller, 2012, Hill et al., 2014). The proportion of aberrantly methylated miRNA gene promoters in cancer cells is several fold higher than that of protein-coding genes, indicating that DNA methylation plays an important role in miRNA deregulation in cancer (Vrba et al., 2013). The last decade saw a sharp increase in the interest in identifying targets of methylation and the importance of miRNAs in various tumors, leading to progress in personalized treatment for multiple cancers, including breast cancer (BC) (Stefansson and Esteller, 2013).

BC is the most common cancer among women in all parts of the world, and it accounts for 1/10 of all malignancies detected in both men and women (Dey, 2014). A total of 1.6 million new BC cases are registered annually, and the incidence of BC is increasing worldwide. BC is also the primary cause of cancer-related death among women globally (Dey, 2014).

The aim of this study was to assess the role of methylation of promoter CpG islands in deregulation of 13 cancer-associated miRNAs (miR-124, -125b, -127, -132, -137, -148a, -191, -193a, -203, -212, -34b, -375, and -9) in BC cells. Previously, it was shown that these miRNAs are involved in pathogenesis of BC. MiR-124, -125b, -127, -132, -137, -203, and -212 were reported to act as tumor-suppressors that could prohibit proliferation, invasion, migration, and metastasis (Lv et al., 2011, Feliciano et al., 2013, Wang et al., 2014, Zhang et al., 2014, Eedunuri et al., 2015, Taipaleenmaki et al., 2015, Yang et al., 2015a). The miR-212/132 cluster was suggested to enhance synergistic anti-metastatic properties by suppressing the oncogene SOX4 (Hanieh, 2015). Most miRNAs listed above were shown to be down-regulated in BC; however, up-regulation was observed for miR-191-5p and miR-375 (Saito and Saito, 2012, Mar-Aguilar et al., 2013). It was also found that miR-375, which is overexpressed in BC cells, may have oncogenic functions and could be a key driver for the proliferation of ERα-positive BC cell lines (de Souza Rocha Simonini et al., 2010). Moreover, miR-375 was suggested to be a predictor of local relapse in early-stage BC and metastasis (Madhavan et al., 2016, Zehentmayr et al., 2016).

All the selected miRNA genes harbor CpG islands in their promoter regions. Previously, the role of methylation in the deregulation of miR-9, -148a, and -124 was investigated in representative sets of primary breast tumors (Hsu et al., 2009, Ben Gacem et al., 2014, Sandhu et al., 2014). However, the methylation status alteration of MIR-127, -132, -193a, -212, and -191 has not been studied in BC, although the importance of methylation of these genes in pathogenesis of tumors has been demonstrated in other cancers (Heller et al., 2012, Formosa et al., 2013). Moreover, crucial alterations have been reported in the expression of most of these miRNAs in BC (Mar-Aguilar et al., 2013, Wang et al., 2014, Damavandi et al., 2016). Previous studies on methylation aberrations in the rest of the miRNA genes (MIR-203, -34b, -125b-1, -137, and -375) were limited to cancer cell lines or very small subsets of primary human specimens, and the link between miRNA expression and gene methylation in a representative set of primary BC samples have not been addressed directly (Vogt et al., 2011, Zhang et al., 2011a, Zhang et al., 2011b, Vrba et al., 2013). Here, the methylation status of promoter CpG islands of 13 miRNA genes was assessed by methylation-specific PCR (MSP) in 58 primary BC samples paired with histologically normal samples. The relative expression of these miRNAs was assayed by quantitative PCR (qPCR) in a subset of 38 paired BC samples. The data obtained allowed us to analyze the correlation between methylation and expression of the selected miRNAs and to speculate on the role of DNA methylation in altering the expression of these miRNAs in BC.

To analyze the functional role of the 13 selected miRNAs, we tested their ability to deregulate five cancer-associated genes, which were predicted to be targets for the majority of these miRNAs using bioinformatics. We chose the well-known tumor-suppressor gene, RASSF1(isoform A), which is involved in cell cycle arrest and apoptosis induction; the pro-apoptotic tumor-suppressors APAF1 and DAPK1; anti-apoptotic BCL2; and CHL1, which is involved in cell adhesion, cell migration, invasion, and cancer progression (http://www.genecards.org). All five genes harbor CpG islands in their promoter regions; methylation studies of these genes in solid tumors, including BC, have been reported by us previously (Dreijerink et al., 2001, Zabarovsky et al., 2011a, Zabarovsky et al., 2011b, Braga et al., 2015, Pronina et al., 2016). Here, alterations in the expression of these five genes were assayed using qPCR in a subset of 41 paired BC samples that allowed us to analyze the correlations between the expression levels of the 13 miRNAs and the 5 protein-coding genes and to speculate on possible miRNA-mRNA interacting pairs in BC.

In addition, we analyzed the association between methylation alterations of the 13 miRNA genes and BC progression and identified miRNA genes which hypermethylation was associated with BC progression.