GES1, an immortalized normal gastric cell line, was obtained from the Oncology Institute of China Medical University (Shenyang, China). Four human GC cell lines, HGC27, AGS, BGC823 and SGC7901 were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). These cells were cultured in RPMI-1640 medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) and incubated at 37°C in a humidified 5% CO₂ atmosphere. Four GC lines were incubated in culture medium with 5 μM DNMT inhibitor 5-aza-2′-de-oxycytidine (DAC; Sigma, St. Louis, MO, USA) for 3 days, and 0.3 μM of the histone deacetylase inhibitor trichostatin A (TSA; Sigma) for 1 day. The time, dose and sequence of DAC and/or TSA were based on previous studies (19,20).

Human GC samples were collected from 40 patients who underwent gastrectomy at Cancer Institute of China Medical University (Shenyang, China) between January 2009 and June 2011. All GC cases were pathologically confirmed. Non-malignant gastric tissues that were ≥5 cm away from the tumor were obtained from the patients. This study was approved by the Institutional Review Board of China Medical University.

Five million cells were crosslinked with 1% formaldehyde for 10 min at 37°C, and then 0.125 M glycine was added to stop the cross-linking. After washing with ice-cold PBS, the cell pellets were resuspended in lysis buffer, and sonicated to generate 200–1,000-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; Millipore, Billerica, MA, USA), Lys-9 acetylated histone H3 (07-352; Millipore) or Lys-4 trimethylated histone H3 (07-472; Millipore) at 4°C overnight. The second lysate was incubated with normal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as a negative control. The third lysate was used as an input control. We added protein G-Sepharose beads 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.

A total of 2 μl of immunoprecipitated DNA, DNA input control and negative control were used for PCR. The following primer set for PCR were designed to amplify the overlapping fragments of 190 bp along the PSD promoter: sense: 5′-gactggctt ctgtc-gtcctc-3′ and antisense: 5′-ggcagacagtaagagcctgg-3′. PCR products were subjected to 2.5% agarose gel electrophoresis at 120 V for 40 min and quantified using the Fluor Chen 2.0 system (Bio-Rad, Hercules, CA, USA). For quantitation, 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 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. The ChIP real-time PCR experiments were repeated three times.

Total RNA was extracted from cells and tissues with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. 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 real-time PCR with the following program: 95°C for 30 sec and 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 each primer, 2 μl cDNA, and 8.5 μl diethylpyrocarbonate (DEPC)-treated water. Primers used were 5′-CTGGGCAAGAACAATGACTTC-3′ (sense) and 5′-GAGGACAGGGCTTCAGGATT-3′ (antisense) for PSD; and 5′-CATGAGAAGTATGACAACAGCCT-3′ (sense) and 5′-AGTCCTTCCACGATACCAAAGT-3′ (antisense) for glyceraldehyde-3-phosphate dehydrogenase (GADPH). The negative control used DEPC-treated water to replace cDNA templates for every PCR. The PSD level was expressed as Ct after normalization to the levels of GAPDH mRNA. The experiment was done in triplicate.

Three pairs of the PSD siRNA sequence were designed and synthesized by GenePharma (Shanghai, China). As a result of relative effectiveness and stability, the following siRNA sequence was selected in our experiments: 5′-CCAAGCUCAGGGUGUUUTT-3′, 5′-AAA CACCCUGAGAGCUUGGTT-3′; it was transfected into SGC7901 cells. A non-silencing siRNA sequence was used as a negative control (5′-UUCUCCGAACGUGUCACGUTT-3′, 5′-ACGUGACACGUUCGGAGAATT-3′). siRNA transfections were performed according to the manufacturer’s instructions. SGC7901 cells were seeded in 6-well plates in medium containing 10% serum 24 h before the experiment. PSD or negative control siRNA of 100 pmol was diluted in 250 μl Opti-MEM I medium. Diluted siRNA was mixed with diluted Lipofectamine 2000 for 20 min. The oligomer-Lipofectamine complexes were applied to the SGC7901 cells. Following transfection, the PSD mRNA levels were assessed 48 h later.

Genomic DNA was extracted from cells and tissues with phenol-chloroform-isoamyl alcohol and collected by ethanol precipitation. The bisulfite treatment was performed using the EZ DNA Methylation-Gold kit (Zymo Research, Los Angeles, CA, USA) according to the manufacturer’s protocol. The CpG map of the PSD promoter and the location of primers used in this study were analyzed by http://www.urogene.org/methprimer/index1.html (21). We found five CpG islands in the PSD promoter region. The primers for the methylated PSD CpG island were 5′-GTTG TAGGGAAGCGGTTC-3′ (sense) and 5′-CGACCACGAAA AAAAACC-3′ (antisense). The primers for the unmethylated PSD CpG islands were 5′-AGGGTTGTAGGGAAGTGG TTT-3′ (sense) and 5′-CAACCACAAAAAAAAACCTA-3′ (antisense). 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.

Total protein was extracted from cells and tissues in a lysate buffer: 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% Nonidet P40, 0.5% sodium deoxycholate and phenylmethylsulfonyl fluoride (all from Beyotime Institute of Biotechnology, Shanghai, China). Each sample (60 μg) was electrophoresed in 10% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). After blocking with 5% BSA in Tris-buffered saline-Tween-20 (20 mM Tris-HCl, 500 mM NaCl, 0.05% Tween-20) for 2 h at room temperature and then incubated with primary antibodies for PSD (1:500 dilution; Novus International, Littleton, MA, USA), caspase-3 (1:500 dilution; Millipore), caspase-7 (1:500 dilution; Millipore) or β-actin (1:2,000 dilution; ZSGB-Bio, Beijing, China) overnight at 4°C. The next day, after incubation with a 1:2,000 dilution of secondary antibodies at 37°C for 2 h, and after washing, the immunoreactive protein bands were visualized using an electrochemiluminescence (ECL) detection kit (Thermol Biotech, Rockford, IL, USA). The ratio between the optical density of the protein of interest and β-actin was calculated as the relative content of the protein detected. Each experiment was repeated three times.

Cells were plated in 6-well plates and maintained in RPMI-1640 medium containing 10% fetal calf serum. A wound was created in the center of the cell monolayer by a sterile plastic pipette tip. The cells were allowed to migrate for 24 h. Images were taken at 0, 12 and 24 h after wounding to assess the ability of the cells to migrate into the wound area using an inverted microscope (IX-71; Olympus, Tokyo, Japan).

Approximately 5×10⁴ cells cultured in 200 μl serum-free RPMI-1640 medium were seeded onto Matrigel-coated 8-μm pore size Transwell filters (Corning Life Sciences, Corning, NY, USA) in the upper chambers. Five hundred microliters of RPMI-1640 containing 10% fetal calf serum was added to the lower chambers as a chemoattractant. Cells were incubated at 37°C in a humidified 5% CO₂ atmosphere for 24 h. Cells that had successfully invaded through the inserts were fixed in 4% paraformaldehyde for 30 min, and stained with methylrosanilinium chloride. The invading cells were counted from five preselected microscopic fields of view at ×200 magnification. The assay was performed from three independent experiments.

Cell proliferation was evaluated by CCK-8 assay (Beyotime Institute of Biotechnology). Cells were seeded in 96-well plates (3×10³ per well). CCK-8 solution (10/100 μl medium) was added to each well, and cells were incubated for 1 h at 37°C. Absorbance was measured at 450 nm using Synergy2 Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA). The assay was conducted in five replicate wells for each sample and three parallel experiments were performed.

All statistical analyses were performed using SPSS version 17.0 (SPSS, Chicago, IL, USA). We used the χ² test and Fisher’s exact-test to analyze the correlations between PSD expression and clinicopathological characteristics. Student’s t-test or one-way ANOVA were used for continuous variables. Data are expressed as mean ± SD. A value of p<0.05 was considered statistically significant.

Using real-time RT-PCR to assess mRNA expression of PSD, we found that PSD was downregulated in HGC27 (0.3651±0.020), AGS (0.4198±0.053), BGC823 (0.467±0.077) and SGC7901 (0.8188±0.009) cells compared to the normal mucosal line, GES1 (1-fold as the control) (p<0.05; Fig. 1A and B). We also analyzed PSD mRNA expression in 40 paired GC specimens and corresponding normal tissues. PSD mRNA expression was significantly lower in GC tissues than in their corresponding normal tissues (0.3406±0.017 vs. 0.5115±0.018), (p<0.01, Fig. 1C and D). Western blotting showed that protein expression of PSD in GC tissues was also lower than in their corresponding non-tumor tissues (0.2970±0.01706 vs. 0.5523±0.02593), (p<0.01, Fig. 2).

We further investigated the relationship between PSD expression and clinicopathological factors. PSD mRNA and PSD protein levels were both related to tumor differentiation and lymph node metastasis (Tables I and II). There was no correlation with any of the clinicopathological features, including age, sex, tumor size, invasion depth and TNM stage.

To elucidate whether the aberrant DNA methylation and histone modification of PSD were associated with loss of PSD, we treated GC cell lines HGC27, AGS, BCG823 and SGC7901 with epigenetic agents (DAC and TSA). DAC and TSA had different effects on PSD expression in the PSD-positive cell line (SGC7901) and PSD-negative cell lines (HGC27, AGS and BCG823). After treatment with DAC or TSA alone, the PSD mRNA was induced in HGC27, AGS and BCG823 cells. Combined treatment with both agents restored PSD expression to a significantly greater degree than did treatment with either agent alone. In SGC7901 cells, treatment with DAC and TSA, alone or in combination, had no significant effect on the restoration of PSD expression (Fig. 3).

To probe the role of histone modifications in the regulation of the PSD gene expression, we examined the histone markers H3-K9 trimethylation, H3-K9 acetylation and H3-K4 trimeth-ylation with the PSD promoter region using ChIP. As shown in Fig. 4, in SGC7901 cells, where PSD was expressed, the level of H3-K9 trimethylation in the promoter regions was minimal. In comparison, H3-K9 trimethylation levels in the PSD gene promoter were high in HGC27, AGS and BCG823 cells. We found that the levels of both H3-K9 acetylation and H3-K4 trimethylation at PSD promoter regions were minimal in HGC27, AGS and BCG823 cell lines. However, these levels were higher in SGC7901 cells. After treatment with DAC, H3-K9 trimethylation in the PSD promoter was decreased significantly and H3-K4 trimethylation was increased significantly in HGC27, AGS and BGC823 cells. We also found that TSA increased H3-K9 acetylation in the PSD promoter, but had no effect on H3-K9 trimethylation and H3-K4 trimethylation in HGC27, AGS and BGC823 cells. Combined treatment significantly decreased H3-K9 trimethylation, and increased H3-K9 acetylation and H3-K4 trimethylation in the PSD promoter in PSD-negative cell lines. The efficiency of combined treatment was similar to DAC or TSA alone. In the PSD-positive cell line (SGC7901), treatment with DAC, TSA or both had no significant effect on the histone markers H3-K9 trimethylation, H3-K9 acetylation and H3-K4 trimethylation.

The PSD promoters contain five CpG islands, so we can suppose that low PSD expression is due to DNA methylation. In order to test this hypothesis, we first examined the DNA methylation of PSD in GC cell lines using MSP. We observed hypermethylation of PSD in HGC27 and AGS cells, partial methylation in BGC823, and no methylation in SGC7901 cells (Fig. 5). In PSD-negative cell lines (HGC27, AGS and BGC823), treatment with DAC resulted in DNA demethylation. TSA had no effect on DNA methylation of PSD, and combined treatment had no additional effect on DNA demethylation beyond that produced by treatment with DAC alone. In unmethylated SGC7901 cells, treatment with DAC, TSA or both did not have a significant effect on DNA methylation. MSP was performed to detect methylation status of PSD gene in 40 paired tumor and corresponding non-malignant gastric tissues. DNA methylation in gastric tissues, which included methylated and partially methylated tissues, occurred in 60% (24/40) of primary GC tissues and 27.5% (11/40) of non-malignant gastric tissues. No methylation was observed in 16 (40%) primary GC tissues and 29 (72.5%) non-malignant gastric tissues. The difference in methylation status of PSD between primary GC and non-malignant gastric tissue specimens was significant (p<0.01, Table III).

To explore further the tumor-suppressive function of PSD, we used PSD-specific siRNA to knock down PSD expression in the SGC7901 cell line, in which the level of PSD was relatively high. RT-PCR confirmed that PSD was downregulated after 48-h transfection (Fig. 6A). Wound-healing assay showed that in SGC7901 cells, knockdown of PSD led to cell migration at 24 h after establishing the wound (Fig. 6B). We also examined whether cell invasive capacity was altered in PSD-depleted cells. Matrigel invasion assay showed that SGC7901 cells transfected with PSD siRNA had more invasion (187.5±4.518) than cells transfected with control siRNA (66.5±1.848) or untreated SGC7901 cells (64.2±1.250) (p<0.01, Fig. 6C).

To evaluate the potential effects of transfection with PSD siRNA on growth of SGC7901 cells, we examined the growth curve by CCK-8 assay. Depletion of PSD promoted growth of SGC7901 cells in a time-dependent manner (Fig. 7A). We then sought to determine the possible mechanisms that underlie the depletion of PSD-promoted growth of SGC7901 cells. Levels of apoptosis-related proteins (caspase-3 and -7) were evaluated using western blot analysis. Caspase-3 and -7 protein levels decreased after 48 h of transfection with PSD siRNA (Fig. 7B).