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Screening of Significantly Hypermethylated Genes in Breast Cancer Using Microarray-Based Methylated-Cpg Island Recovery Assay An INTERNATIONAL JOURNAL OF ONCOLOGY 41: 629-638, 2012 Screening of significantly hypermethylated genes in breast cancer using microarray-based methylated-CpG island recovery assay and identification of their expression levels ZHEN-QIANG LIAN, QI WANG, WEN-PING LI, AN-QIN ZHANG and LING WU Breast Disease Center, Guangdong Women and Children Hospital of Guangzhou Medical College, Guangzhou 510010, P.R. China Received February 10, 2012; Accepted April 17, 2012 DOI: 10.3892/ijo.2012.1464 Abstract. To screen candidate methylation markers for early of the relationship between methylation and gene expression. detection of breast cancer and to explore the relationship The MIRA approach can screen candidate methylated genes between methylation and gene expression, we performed for further clinical validation more effectively than gene methylated-CpG island recovery assay (MIRA) combined expression microarray-based strategy. with CpG island array on 61982 CpG sites across 4162 genes in 10 cancerous and 10 non-cancerous breast tissues. Direct Introduction bisulfite sequencing and combined bisulfite restriction analysis (COBRA) were carried out in independent cancerous With an estimated 1.38 million new cancer cases worldwide and non-cancerous samples. Gene expression was analyzed in 2008, breast cancer is the most frequent cancer and the by microarrays and validated using RT-PCR. We detected 70 leading cause of cancer mortality in women (1). If breast significantly hypermethylated genes in breast cancer tissues, cancer is detected and treated at an early stage, the majority including many novel hypermethylated genes such as ITGA4, of the patients survive. These facts highlight an urgent need to NFIX, OTX2 and FGF12. Direct bisulfite sequencing showed develop novel molecular biomarkers that can detect early stage widespread methylation occurring in intragenic regions of the of breast cancer so that treatment is initiated promptly. WT1, PAX6 and ITGA4 genes and in the promoter region of the Carcinogenesis has been demonstrated to involve not only OTX2 gene in breast cancer tissues. COBRA assay confirmed genetic but also epigenetic alterations that may suppress or that the WT1, OTX2 and PAX6 genes were hypermethylated in activate multiple genes. The most common epigenetic altera- breast cancer tissues. Clustering analysis of the gene expression tion in cancer is DNA methylation (2), which is an early event of 70 significantly hypermethylated genes revealed that most in carcinogenesis, and may therefore serve as a biomarker hypermethylated genes in breast cancer were not expressed in for the early detection of cancer. Promoter methylation, with breast tissues. RT-PCR assay confirmed that WT1 and PITX2 a methyl group to the 5-carbon position of cytosine in CpG were only weakly expressed in the breast cancer tissues and islands, is associated with transcriptional silencing. DNA were not expressed in most non-cancerous breast tissues. OTX2 methylation also occurs in the intragenic region. To date, little and PAX6 were not expressed in either breast cancer or non- attention has been paid to intragenic methylation. cancerous tissues. In conclusion, these results will expand our Recently, aberrant methylations of specific genes that knowledge of hypermethylated genes and methylation sites for involve breast carcinogenesis have been widely explored and early detection of breast cancer and deepen our understanding the list of aberrant methylated genes is increasing (3,4). Due to the limitation of techniques for analysis of DNA methylation, the majority of these efforts have focused only on individual candidate genes, which have already been identified to play Correspondence to: Professor Qi Wang, Breast Disease Center, a role in other cancer models. These genes are mostly tumor Guangdong Women and Children Hospital of Guangzhou Medical suppressor genes. Thus far, most methylated genes that College, Guangzhou, Guangdong 510010, P.R. China contribute to breast cancer, such as methylated p16, CCDN2, E-mail: [email protected] MGMT and hMTLH1 are identified by the candidate approach, confining to promoter methylation of tumor suppressor Abbreviations: COBRA, combined bisulfite restriction analysis; genes (5). This approach is not only time-consuming and RT-PCR, reverse transcription PCR; MIRA, methylated-CpG island recovery assay labor-intensive, but also limited by candidate methylated genes and candidate methylated sites available. Key words: breast cancer, CpG island methylation, Methyl-CpG Gene expression microarray based strategy has been binding protein, microarray, gene expression, combined bisulfite developed for high-throughput screening of DNA meth- restriction analysis ylation biomarker on a genomic scale in breast cancer. This approach uses microarray to interrogate upregulated genes 630 LIAN et al: HYPERMETHYLATED GENES AND THEIR EXPRESSION LEVELS IN BREAST CANCER IDENTIFIED BY MICROARRAY after treatment of breast cancer line with a demethylating Table I. Clinicopathological characteristics of the patient agent 5-aza-2'-deoxycytidine (6), and then uses bisulfite samples. sequencing, restriction analysis, and/or methylation specific PCR to confirm DNA methylation. This approach might leave Characteristics Breast cancer Breast non-cancer out non-expressed genes in the target tissue, thereby missing tissues (n=15) tissues (n=15) candidate genes, which may actually be potential methylation biomarkers in cancer diagnosis. For example, the significantly Mean age (y) (range) 41 (34-50) 38 (32-47) methylated vimentin gene has been used as a diagnostic Histological type Invasive ductal No histological biomarker in colorectal cancer, while colon cancer cells do not carcinoma findings express vimentin (7,8). Using the gene expression microarray Grade (%) ND based strategy, methylation of vimentin could not be identified. I 5 (33.3%) However, it is unclear whether there are that many such non- II 10 (66.7%) expressed methylated genes in cancerous and non-cancerous Cancer size (%) ND tissue that critical methylation biomarkers could not be identi- pT1 8 (53.3%) pT2 7 (46.7%) fied by gene expression microarray based strategy. Recently, methyl-DNA immunoprecipitation combined Lymph node status (%) ND Negative 15 (100.0%) with high-throughput sequencing (9) and methylated-CpG Positive 0 (0.0%) island recovery assay (MIRA) combined with CpG island Estrogen receptor (%) ND array (10,11) were also used to screen cancer-associated Positive 8 (53.3%) methylated genes in clinical samples of breast cancer tissue. Negative 7 (46.7%) To screen novel biomarkers for the early detection of breast Progesterone receptor (%) ND cancer, in the current study, we utilized a high-throughput Positive 9 (53.3%) genome-wide approach, MIRA microarray, that is based on the Negative 6 (46.7%) high affinity of the MBD protein for methylated DNA to iden- HER2 status (%) ND tify novel tumor-specific genes with promoter methylation or (-)/ (+) 10 (66.7%) intragenic methylation. We also integrated the corresponding (++) 3 (20.0%) gene expression profile to explore the role of methylation in (+++) 2 (13.3%) regulating gene expression. Materials and methods 200-1000 bp fragments. Fragment sizes were verified by gel Patient samples. Samples of breast cancer tissue were obtained electrophoresis in 1.5% agarose gels. To remove fragments from the surgical specimens of 15 patients with early stage smaller than 100 bp, the fragmented DNA was purified using invasive breast cancer. The tissue sections were stained with the MicroDNA Purification Kit (Beijing CoWin Biotech Co. H&E and were reviewed by a pathologist to confirm the pres- Ltd, Beijing, China) following the manufacturer's instructions. ence of the cancer lesions. Breast cancer was staged according Subsequently, the purified DNA fragments were used to enrich to the American Joint Committee on Cancer staging system methylated DNA using the BioChain MBD kit (BioChain, protocol. ER, PR and HER2 status were determined by immu- Hayward, CA, USA) according to the manufacturer's protocol. nohistochemistry. Non-cancerous breast tissues were collected This kit uses the high affinity of the MBD protein for double- from the biopsy specimens of 15 patients for the suspicion stranded CpG-methylation DNA. This MBD protein can bind of malignant lesion but without any histological findings. methylated DNA fragments more sensitively and specifically Clinicopathological characteristics of the patient samples are than antibody, and thus has a low false positive rate in the given in Table Ι. Ten cancerous and 10 non-cancerous breast enrichment of methylated DNA fragments (12). In addition, its tissues were selected for microarray analysis and the other procedure is simple since MBDs can bind dsDNA (12), natural 5 cancerous and 5 non-cancerous breast tissues were used for status of genomic DNA, while antibody can bind only ssDNA validation of the genes. All samples were snap-frozen in liquid and therefore has to denature the dsDNA before DNA enrich- nitrogen and stored at -80˚C until nucleic acid extraction. The ment in antibody based methods. study was approved by the Ethics Committee of our hospital, The MIRA-captured DNA segments were purified using and all patients provided written informed consent. the MicroDNA Purification Kit (CoWin Biotech Co.). After purification, the MIRA-captured DNA segments were ampli- MIRA microarray analysis of DNA methylation. The fied using the GenomePlex Whole Genome Amplification Kit MIRA microarray analysis was
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