ONCOLOGY REPORTS 29: 1819-1826, 2013 Genome-wide analysis of DNA methylation in tongue squamous cell carcinoma SHENG ZHANG1, XIANG-LING FENG2, LEI SHI3, CHAO-JIAN GONG1, ZHI-JING HE1, HAN-JIANG WU1 and TIAN-YOU LING1 1Department of Oral and Maxillofacial Surgery, the Second Xiangya Hospital, Central South University, Changsha, Hunan 410011; 2Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, Hunan 410008; 3Department of Pathology, the Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, P.R. China Received December 29, 2012; Accepted January 23, 2013 DOI: 10.3892/or.2013.2309 Abstract. Tongue squamous cell carcinoma (TSCC) is one of Introduction the most common types of oral cancer; however, its molecular mechanisms remain unclear. In this study, methylated DNA Oral cancer accounts for 2-4% of all cancer cases world- immunoprecipitation (MeDIP) coupled with methylation wide (1). An estimated 263,900 new cases and 128,000 deaths microarray analysis was performed to screen for aberrantly from oral cancer occurred globally in 2008 (2). More than methylated genes in adjacent normal control and TSCC tissues 90% of all oral cancer cases are oral squamous cell carci- from 9 patients. Roche NimbleGen Human DNA Methylation nomas (OSCCs), which are a result of multistep processes that 385K Promoter Plus CpG Island Arrays were used to detect develop from the combined effects of a patient's genetic predis- 28,226 CpG sites. A total of 1,269 hypermethylated CpG position and exposure to environmental influences, including sites covering 330 genes and 1,385 hypomethylated CpG sites tobacco, alcohol, betel quid, chronic inflammation and viral covering 321 genes were found in TSCC tissue, compared to the infection (3). Tongue squamous cell carcinoma (TSCC) is a adjacent normal tissue. Furthermore, we chose three candidate common OSCC. Imaging systems and treatment strategies in genes (FBLN1, ITIH5 and RUNX3) and validated the DNA TSCC are improving substantially, however, 5-year survival methylation status by methylation-specific PCR (MS-PCR) statistics remain low. The molecular mechanisms underlying and the mRNA expression levels by reverse transcription PCR its clinical characteristics including carcinogenesis, develop- (RT-PCR). In TSCC tissue, FBLN1 and ITIH5 were shown ment, progression, invasion, and metastasis have yet to be fully to be hypermethylated and their expression was found to be elucidated. Epigenetics studies recently showed that aberrant decreased, and RUNX3 was shown to be hypomethylated, DNA methylation is involved in the progress of TSCC (4). however, its mRNA expression was found to be increased. DNA methylation is an important regulator of gene transcrip- In addition, another three genes (BCL2L14, CDCP1 and tion and its role in carcinogenesis and development has been DIRAS3) were tested by RT-PCR. In TSCC tissue, BCL2L14 a topic of considerable interest in recent years. DNA methyla- and CDCP1 expressions were markedly upregulated, and tion, which represses transcription of the promoter region of DIRAS3 expression was significantly downregulated. Our data tumor suppressor genes leading to gene silencing, has been demonstrated that aberrant DNA methylation is observed in studied extensively (5). TSCC tissue and plays an important role in the tumorigenesis, DNA methylation is a very stable epigenetic mark and next development and progression of TSCC. generation sequencing studies have shown that several genes are aberrantly methylated in various types of cancer (6,7). Tissue specific DNA methylation patterns are stabilized during embryonic development, and are maintained through cell divisions. Aberrant DNA methylation patterns have been associated with a large number of human malignancies and are Correspondence to: Dr Sheng Zhang, Department of Oral and found in two distinct forms: hypermethylation and hypometh- Maxillofacial Surgery, the Second Xiangya Hospital, Central South ylation (8). The majority of DNA methylation studies focus University, 139 Middle Renmin Road, Changsha, Hunan 410011, on the analysis of CpG islands located in the promoter areas P. R. Ch i na E-mail: [email protected] of candidate genes. However, differentially methylated areas may be located within genes and at large distances from the Key words: tongue squamous cell carcinoma, DNA methylation, nearest neighboring genes (9). In the past decade, researchers CpG island, microarray, biomarker focused only on a few candidate genes of DNA methylation in TSCC-related genes (4,5,10). Nevertheless, systematic studies on the genome-wide distribution of methylated loci in TSCC 1820 ZHANG et al: GENOME-WIDE METHYLATION ANALYSIS IN TSCC Table I. List of primers of methylated (M) and unmethylated (U) sequences for MS-PCR analysis. Gene Primer Sequence FBLN1 M primer F: 5'-ATTAGGAGATTCGCGGTTTC-3' R: 5'-GCTCCATAAACGACGAACG-3' U primer F: 5'-GATTAGGAGATTTGTGGTTTTG-3' R: 5'-CACACTCCATAAACAACAAACA-3' ITIH5 M primer F: 5'-TTGGCGATAGAAATTAAGTAAGTTC-3' R: 5'-AACCACCTATATTAACCCACG-3' U primer F: 5'-TTGGTGATAGAAATTAAGTAAGTTTGT-3' R: 5'-AAAACCACCTATATTAACCCCACA-3' RUNX3 M primer F: 5'-TTACGAGGGGCGGTCGTACGCGGG-3' R: 5'-AAAACGACCGACGCGAACGCCTCC-3' U primer F: 5'-TTATGAGGGGTGGTTGTATGTGGG-3' R: 5'-AAAACAACCAACACAAACACCTCC-3' remain scarce. With the advent of next generation sequencing, methylated DNA was obtained by whole-genome amplifica- genome-wide screening has become an attractive and useful tion using the GenomePlex® Whole Genome Amplification kit tool for profiling TSCC. (Sigma-Aldrich, St. Louis, MO, USA). MeDIP DNA and input In this study, methylated DNA immunoprecipitation DNA were labeled with Cy5 and Cy3, respectively, and then (MeDIP) in combination with microarray-based hybridization analyzed using Human DNA Methylation 385K Promoter was performed in adjacent normal control and tumor tissues Plus CpG Island Arrays (Roche NimbleGen, Madison, WI, from 9 patients with TSCC (11). Extensive DNA methylation USA), which were used to detect 28,226 CpG sites. Gene distribution information from human TSCC and its adjacent chips were scanned by GenePix 4000B microarray scanner normal tissues provided a preliminary genomic DNA meth- and hybridization signals were analyzed using GenePix Pro ylation profile. Selected differentially expressed genes were 6.0 hybridization analysis software. Probe-rich value setting further validated using methylation-specific PCR (MS-PCR) was ≥2.0. and reverse transcription PCR (RT-PCR). MS-PCR analysis. To determine the methylation status of the Materials and methods three selected genes (FBLN1, ITIH5 and RUNX3), we used MS-PCR in 20 pairs of samples from adjacent normal control Patients and samples. Samples were obtained from 20 patients and TSCC tissues. Genomic DNA was isolated by using diagnosed with TSCC who underwent surgery at the Second Wizard® Genomic DNA Purification kit (Promega, Madison, Xiangya Hospital of Central South University and the Hunan WI, USA). Complete bisulfite conversion and purification of Provincial Tumor Hospital. The study protocol was approved DNA were performed according to the EpiTect® Bisulfite kit by the Ethics Committees of Central South University and (Qiagen). The fully methylated and fully unmethylated DNA written informed consent was obtained from all patients. None samples were used as controls, and a water blank reaction of the patients were given adjuvant chemotherapy or radiation was used as control for contamination. Following amplifi- prior to the operation. For each TSCC case, fresh samples cation by PCR, products were resolved on 1% agarose gels were obtained from the primary tumor tissue and the adjacent containing 0.5 µg/ml ethidium bromide and visualized under normal tongue mucosa with a clear surgical margin to tumor. UV transillumination. Primers for MS-PCR analysis are Surgical margins were considered as tumor-free or negative shown in Table I. at least 5 mm of histologically normal tissue according to the pathological examination. The samples were snap frozen in RT-PCR analysis. To determine the mRNA expression level liquid nitrogen and stored at -80˚C until subsequent analyses. of the six selected genes (FBLN1, ITIH5, RUNX3, BCL2L14, CDCP1 and DIRAS3), we used RT-PCR in 20 pairs of samples MeDIP and methylation microarray hybridization. Genomic from adjacent normal control and TSCC tissues. Total RNA DNA from TSCC and adjacent normal control tissue (n=9) was isolated from the sample tissues using TRIzol® reagent were extracted and purified according to the DNeasy Blood (Invitrogen, Grand Island, NY, USA) according to the manu- and Tissue kit (Qiagen, Germantown, MD, USA). Then, DNA facturer's protocol. RNA was efficiently reverse transcribed samples were pooled to one pair and sheared by sonication to into cDNA using the Reverse Transcription System (Promega). obtain fragments between 400 and 500 bp and were immuno- Primers for RT-PCR analysis and the PCR conditions of the precipitated by anti-5-methylcytidine antibody according six selected genes are shown in Table II. The expression level to the MagMeDIP kit (Diagenode, Liège, Belgium). Fully of each gene was confirmed by comparison to the expres- ONCOLOGY REPORTS 29: 1819-1826, 2013 1821 Table II. List of primers for RT-PCR analysis and the condition of PCR. Gene Primer Size of Annealing Cycles product (bp) temperature (˚C) FBLN1 F: 5'-TCAATACAGTTGCCTAGAGCA-3' 409 60 28 R: 5'-GAGGAACAAGAGGACCCAT-3' ITIH5 F: 5'-CACAGTTCTCCAGCGACA-3' 386 60 32 R: 5'-CAGGACCGTTTCAGTATCAT-3'