The human MKN45, AGS, BGC823 and SGC7901 GC cell lines were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The immortalized GES1 normal gastric cell line was obtained from the oncology Institute of China Medical University. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco-BRL, Grand Island, NY, USA) and incubated at 37°C in a humidified 5% Co₂ atmosphere. The four GC lines were incubated in culture media with 5 μM of the DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (DAC) for 3 days, and 0.3 μM of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) (both from Sigma-Aldrich, St. Louis, MO, USA) for 1 day. The time, dose and sequence of DAC and/or TSA were based on previous studies (11,12).

Five million cells were crosslinked with 1% formaldehyde for 10 min at 37°C, and then 0.125 M of glycine was added to stop the crosslinking. After washing with ice-cold PBS, the cell pellets were resuspended in lysis buffer, and sonicated to generate 200 to 1,000-bp DNA fragments. The lysate was then divided into three fractions. The first lysate was precipitated using antibodies against Lys-9 trimethylated histone H3 (05-1242), Lys-9 acetylated histone H3 (07-352) and Lys-4 trimethylated histone H3 (07-472) (all from Millipore, Billerica, MA, USA) at 4°C overnight. The second lysate was incubated with normal rabbit IgG (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) as a negative control. The third lysate was used as an input control. Protein G-Sepharose beads were added to collect the immunoprecipitated complexes and left them to incubate for 1 h at 4°C. After washing, the beads were treated with RNase (50 mg/ml) for 30 min at 37°C and then proteinase K overnight. The crosslinks were then reversed by heating the sample at 65°C for 6 h. DNA was extracted by the phenol/chloroform method, ethanol precipitated, and resuspended in 20 μl water.

Immunoaffinity-enriched DNA fragments (IP) and input samples were amplified using a Whole Genome Amplification kit (Sigma-Aldrich). Input and IP samples were labeled in separate reactions with Cy3 and Cy5, respectively and were co-hybridized to a Human 3 × 720K RefSeq Promoter Array (NimbleGen, Madison, WI, USA), containing 22,542 human promoter regions that cover a range from −3,200 to +800 bp, relative to the transcription start site. The hybridized microarray slides were scanned using a GenePix 4000B scanner (Axon Instruments, Foster City, CA, USA). Data were extracted using NimbleScan software. From the normalized log2-ratio data, a permutation-based peak finding algorithm provided by NimbleScan version 2.3 (NimbleGen) was used to detect peaks that represented significant positive enrichment. To detect the peaks for histone modification, only the false discovery rate (FDR <0.05) was considered for analysis.

ChIP was conducted in the same manner as for ChIP-chip. Briefly, immunoaffinity-enriched, input, and negative control DNA were used for PCR. Primers were designed based on the promoter structure of the genes selected for evaluating ChIP-chip data (Table I). PCR products were visualized on a 2.5% agarose gel. For quantification, PCR amplification was performed on an ABI 7700 real-time PCR (Applied Biosystems, Foster City, CA, USA). PCR conditions included an initial denaturation step of 4 min at 95°C, followed by 35 cycles of 5 sec at 95°C, 30 sec at 59°(60°)C and 20 sec at 72°C. Quantitative ChIP-PCR values were normalized against values from a standard curve constructed using input DNA that was extracted for the ChIP experiment. ChIP experiments were repeated three times for each target gene.

Total RNA was extracted from cells and tissues with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The quality and concentration of RNA were measured by ultraviolet absorbance at 260 and 280 nm (A260/A280 ratio) and checked by agarose gel electrophoresis individually. Total RNA was reverse transcribed into cDNA using an Expand Reverse Transcriptase kit (Takara, Dalian, China). Expression of PSD mRNA was detected using qPCR under the following conditions: 95°C for 30 sec, 35 cycles of 95°C for 5 sec and 60°C for 30 sec. The reaction mixture contained 12.5 μl SYBR-Green (Takara), 1 μl of each primer, 2 μl cDNA, and 8.5 μl diethylpyrocarbonate (DEPC)-treated water. The PCR primers used for each gene in this analysis are shown in Table II. Negative control used DEPC-treated water to replace cDNA templates for each PCR. The PSD level was expressed as C_t after normalization to the levels of GAPDH mRNA. The experiment was performed in triplicate.

Genomic DNA was extracted from cells with phenolchloroform-isoamyl alcohol and collected by ethanol precipitation. Genomic DNA (2 μg) was treated with NaOH (2 M) at 42°C for 20 min. After denaturation, the DNA was incubated with hydroquinone and sodium bisulfate at 54°C for 16 h in the dark. DNA was purified using a DNA Cleanup kit (Promega, Madison, WI, USA), followed by incubation with 3 M NaOH at 37°C for 15 min, and precipitation with ammonium acetate and 100% ethanol at −20°C overnight. The following day, DNA was washed with 70% ethanol, and dissolved in 15 μl TE buffer. The primers used for MSP were located in the promoter region of the genes (Table III). Peripheral blood cell DNA from healthy adults treated with SssI methyltransferase (New England Biolabs, Ipswich, MA, USA) and untreated DNA were used as positive and negative controls, respectively. PCR products were separated by electrophoresis on 2% agarose gels, and quantified using the Fluor Chen 2.0 system.

Statistical analyses were performed using SPSS version 17.0 (SPSS, Chicago, IL, USA). Quantitative data are shown as mean values ± SD. Statistical analyses were performed using the independent sample t-test. P<0.05 was considered statistically significant.

TSG inactivation resulting from higher H3-K9 trimethylation, lower H3-K9 acetylation and H3-K4 trimethylation has been reported to be involved in carcinogenesis. We aimed to identify genes that underwent similar histone modifications in GC cells. We used a two-step strategy to analyze the data. First, we selected 134 candidate genes that showed a high ratio of H3-K9 trimethylation over acetylation (H3-K9Me3/H3-K9Ac >2.0). The selected 20 genes with a high ratio of H3-K9 trimethylation over H3-K9 acetylation are presented in Table IV. We then analyzed 46 candidate genes that showed a high ratio of H3-K9 trimethylation over H3-K4 trimethylation (H3-K9Me3/H3-K4Me3 >2.0). The selected 20 genes with a high ratio of H3-K9 trimethylation over H3-K4 trimethylation are presented in Table V.

To validate the microarray results, the selected genes, PSD, NLK, SMARCC1 and Vps37A exhibited a high ratio of H3-K9 trimethylation over acetylation or a high ratio of H3-K9 methylation over H3-K4 trimethylation in the MKN45 cell line. These results were verified by ChIP-qPCR. As shown in Table VI and Fig. 1, qPCR results for the selected candidate genes were consistent with the ChIP-chip data.

Using qRT-PCR to assess the mRNA expression levels of SMARCC1 and Vps37A, we found that SMARCC1 was downregulated in MKN45 (0.5377±0.01126), AGS (0.5697±0.00963), BGC823 (0.3157±0.0099) and SGC7901 (0.527±0.0046) cells compared to the normal mucosa cell line, GES-1 (1-fold, as was the control). Of note, the mRNA expression levels of Vps37A were similar between GC and GES-1 cells (Fig. 2).

To determine whether epigenetic agents altered PSD, SMARCC1 and Vps37A gene expression levels, we treated MKN45 cells with the DNMT inhibitor DAC, the HDAC inhibitor TSA, or a combined treatment of the two agents. Using RT-qPCR, we found that DAC and TSA had different effects on the expression of these genes in MKN45 cells. DAC alone restored PSD expression, whereas TSA had no effect on PSD expression. The combined treatment restored PSD expression levels similar to those observed after DAC treatment. DAC or TSA alone restored SMARCC1 expression and the combined treatment with the two agents restored SMARCC1 expression to a significantly greater degree than the treatment with either agent alone. Treatment with DAC and TSA, alone or in combination, had no significant effect on Vps37A expression (Fig. 3).

To determine whether DNA methylation affected the expression levels of these genes, we assessed their DNA methylation status in MKN45 cell lines using MSP. We observed hypermethylation of the PSD promoter, partial methylation of the SMARCC1 promoter, and no methylation of the Vps37A promoter, suggesting that the methylation status of these gene promoters was partially responsible for the mRNA expression levels. In MKN45 cells, treatment with DAC resulted in demethylation of the PSD and SMARCC1 promoters, and treatment with TSA resulted in demethylation of the SMARCC1 promoter. However, TSA had no effect on the PSD promoter. Treatment with DAC, TSA or both had no effect on the Vps37A promoter (Fig. 4).