DNA methylation status of REIC/Dkk-3 gene in human malignancies

Surgically resected specimens of 37 primary breast cancers, 42 primary NSCLCs, 21 primary gastric cancers, 20 primary colon cancers, and 7 MPMs were obtained from Okayama University Hospital (Okayama, Japan), 6 MPMs were obtained from Okayama Rousai Hospital (Okayama, Japan), 5 MPMs were obtained from National Sanyo Hospital (Yamaguchi, Japan), and 27 MPMs were obtained from Karmanos Cancer Center (MI). Ten corresponding non-malignant breast tissues and 10 non-malignant lung tissues were also examined. All tissues were frozen with the liquid nitrogen immediately after surgery and were stored at −80°C until extraction of DNA. Institutional Review Board permission and informed consent were obtained for all cases.

Seven breast cancer cell lines (HCC70, HCC1599, HCC1806, MDA-MB-231, MDA-MB-361, MCF7, and ZR75-1), 11 lung cancer cell lines (NCI-H23, NCI-H44, NCI-H125, NCI-H157, NCI-H1299, NCI-H1819, NCI-H1963, NCI-H1975, NCI-H2009, NCI-H358, and A549), 4 MPM cell lines (NCI-H2052, NCI-H2373, NCI-H2452, and NCI-H290), and 6 prostate cancer cell lines (PC3, LNCap-FGC, Du145, Caki-1, Caki-2, and KPK) were examined in this study. MCF7, ZR-75-1, MDA-MB-231, and MDA-MB-361 were obtained from Cell Resource Center for Biomedical Research Institute of Development Aging and Cancer Tohoku University (Miyagi, Japan). Seven cell lines (HCC70, HCC1599, HCC1806, H2052, H2373, H290, and H2452) were kind gifts from Adi F. Gazdar (Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX). Six cell lines (PC3, LNCap-FGC, Du145, Caki-1, Caki-2, and KPK) were kind gifts from the department of urology (Okayama University, Okayama, Japan). The other cell lines were obtained from American Type Culture Collection (Manassas, VA). The cells were maintained in RPMI-1640 medium (Sigma Chemical Co., Saint Louis, MO) supplemented with 10% FBS and were incubated in 5% CO₂.

Genomic DNA was extracted from the surgically resected frozen samples and cultured cells by digestion with SDS/proteinase K followed by phenol/chloroform (1:1) extraction and ethanol precipitation. Two micrograms of each DNA was treated with EZ DNA Methylation Kit (ZYMO RESEARCH, Orange, CA), following the manufacturer’s instructions, and was stored at −20°C until use.

Nested PCR was carried out using bisulfite-treated DNA followed by the restriction enzyme digestion. First-round touchdown PCR was performed under the following conditions: 95°C for 12 min, 40 cycles of 94°C for 45 s, annealing temperature between 58 and 56°C for 1 min, 72°C for 3 min, followed by final extension step at 72°C for 7 min in a 25-μl reaction mixture containing 67 mM Tris–HCl (pH 8.8), 16.6 mM (NH₄)₂SO₄, 6.7 mm MgCl₂, 10 mM β-mercaptoethanol, 1.25 mM of each deoxynucleotide triphosphate (dNTP) mixture, 0.5 μM of each primer, 0.5 unit of HotStar Taq DNA Polymerase (Qiagen, Valencia, CA), and 100 ng of bisulfite-treated DNA. Second-round touchdown PCR was performed using 0.4 μl of the first-round PCR products as a template under same condition, but 47 cycles. Universal methylated DNA and universal unmethylated DNA were used for positive control and negative control, respectively. The location of the CpG dinucleotides in the exon1 and in the 5′-flanking region of REIC/Dkk-3 is shown in Fig. 1. Primers were designed using Primer Express software ver.1.0 in the promoter region of REIC/Dkk-3 type-a. Primers for the first-round PCR were REIC-COBRA-F1 5′-TGGGTTGTTGTAAGTTTGAAGGT-3′ and REIC-COBRA-R1 5′-CTCACCCACCCCRACTAAAC-3′. Primers for the second-round PCR were as follows: REIC-COBRA-F2 5′-TGAAGGTTAGATAAGAYGGGTTTAGG-3′ and REIC-COBRA-R2 5′-ACCCACCCCRACTAAACCRAAT-3′. These primers were designed to ensure amplification of both methylated and unmethylated forms. Two microliters of second PCR products were digested with 3 units of BstUI (whose restriction site is CGCG) for the restriction fragment length polymorphism analysis. The amplicon of second PCR was named RRCOBRA (Region for REIC-COBRA), and the 5 restriction sites of BstUI are shown in Fig. 1. The digested PCR products were visualized on 3% agarose gels stained with ethidium bromide. The percentages of digested band were analyzed by NIH ImageJ 1.37 V software (http://​rsb.​info.​nih.​gov/​ij) as described previously (Xiong and Laird 1997). We performed linear regression analysis of qCOBRA with nested PCR using serial dilution to examine whether qCOBRA with nested PCR really reflected % methylation. We diluted unmethylated DNA amplicon with methylated amplicon to make serial dilution (% methylated DNA; 0, 10, 20, 30, 50, 70, 80, 90, 100%) and performed qCOBRA, as described above.

RRCOBRA was cloned into pCR2.1-TOPO Vector using TOPO TA cloning kit (Invitrogen Life Technologies, Carlsbad, CA) following manufacturer’s instructions. To determine the methylation status in the promoter lesion of REIC/Dkk-3 gene, five breast cancer cell lines (MCF-7, MDA-MB-231, ZR75-1, HCC1806, and HCC1599) and a lung cancer cell line (H1299) were examined. Seven individual clones from each cell line were sequenced using the dGTP BigDye terminator v3.1 Cycle Sequencing Kit with the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA).

Total RNA was extracted from cultured cells using RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s instruction. Oligo(dT)-primed cDNA was synthesized using Super-Script II (Qiagen, Valencia, CA) with DNase treatment. RT-PCR was carried out in 20 μl of reaction mixture with 1xPCR buffer, 200 μM of dNTP, 0.3 μM of each primer, 0.5 units of HotStarTag DNA Polymerase, and 100 ng of cDNA. A touchdown PCR was performed for REIC/Dkk-3 type-a and type-b under the following conditions: 95°C for 12 min, 35 cycles of 94°C for 30 s, annealing temperature between 62 and 58°C for 1 min, 72°C for 3 min, followed by final extension step at 72°C for 7 min. As an internal control, RT-PCR for GAPDH was carried out under the following conditions: 95°C for 12 min, 35 cycles of 94°C for 45 s, 55°C for 90 s, 72°C for 90 s, followed by final extension step at 72°C for 7 min. The primers for REIC/Dkk-3 type-a expression were REIC(a)-F 5′-GGGAGCGAGCAGATCCAGT-3′ (exon1a) and REIC(a)-R 5′-TTTGTCCAGTCTGGTTGTTGGT-3′ (exon3). The primers for REIC/Dkk-3 type-b expression were REIC(b)-F 5′-TGGGAGCTATTAGCGTAGAGGAT-3′ (exon1b) and REIC(b)-R 5′-CATTGTGATAGCTGGGAGGTAAG-3′ (exon3). The PCR products were visualized on 2% agarose gels stained with ethidium bromide. The bands were analyzed using NIH ImageJ 1.37 V software. The expression ratio in each cell line was defined as the ratio of particular sample when compared to those of H1299. To confirm the responsibility of DNA methylation for REIC/Dkk-3 silencing, we treated heavily methylated cell lines (MDA-MB-231) with 5-aza-2′-deoxycytidine (5-Aza-CdR) at the concentration of 5 and 8 μM for 6 days with medium changes on days 1, 3, and 5. Treated and untreated cells from individual triplicate flasks were harvested on day 6 to determine the REIC/Dkk-3 type-a expression using RT-PCR.

Estrogen receptor (ER), progesterone receptor (PgR), and HER2 status in primary breast cancers were obtained from patient medical records. HER2 positive was defined as a score of 2+ and 3+ by immunohistochemistry.

The frequencies of REIC/Dkk-3 methylation between two groups were compared using the Fisher’s exact test or Mann–Whitney’s U test when appropriate. Probability value less than 0.05 was defined as being statistically significant. All data were analyzed by JMP9 for Windows (SAS Institute, Cary, NC).