Six gastric cancer cell lines (MKN-28, AGS, MKN-1, SGC-7901, SNU-5 and KATO III) and one normal gastric epithelial cell line (GES-1) (American Type Culture Collection, Manassas, VA, USA) were used in the present study. The MKN-28 cells were checked for contamination. All cell lines were cultured and maintained in RPMI-1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS) (Gibco Life Technologies, Carlsbad, CA, USA) and 1% penicillin/streptomycin (100 U/ml penicillin and 100 µg/ml streptomycin; Invitrogen Life Technologies, Carlsbad, CA, USA) and incubated in 5% CO₂ at 37°C.

Tumor and paratumor normal tissue samples of 64 patients with GC, who underwent radical gastrectomy at the Affiliated Hospital of Qingdao University (Qingdao, China), were collected between January 2008 and October 2011, and stored in liquid nitrogen. Written informed consent was obtained from all patients and the procedure of the present study was approved by the institutional review board of the Affiliated Hospital of Qingdao University. All samples were confirmed by histology and 36 diffuse-type and 28 intestinal-type samples, as well as their paired paratumor normal tissues, were selected for analysis. Clinicopathological features of patients enrolled in the present study are presented in Table I.

Genomic DNA extraction was conducted with the DNAeasy® Blood & Tissue kit (Qiagen GmbH, Hilden, Germany). RNAiso Plus Reagent (Takara Bio, Inc., Otsu, Japan) was used to extract total RNA, which was utilized in the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) prior to DNA isolation. For RT-qPCR, RNA was reverse transcribed using PrimeScript RT reagent (Takara Bio, Inc.) and the reaction product was treated with RNAse-free DNase I. Details of the RT-qPCR reaction are described below.

A total of 1 µg genomic DNA was subjected to bisulfate treatment using an Epitect Bisulfate kit (Qiagen GmbH). The bisulfate-treated DNA was amplified by qMSP using a LightCycler480 (Roche Diagnostics Ltd., Rotkreuz, Switzerland). Briefly, thermocycling was conducted in a final volume of 25 µl, containing 1.0 µl treated DNA sample, 100 nM of each primer and 12.5 µl SYBR Premix Ex Taq II (Takara Bio, Inc.). The PCR primer sequences for vimentin (GeneChem, Shanghai, China) were as follows: Methylated sense, 5′-TCGTTTCGAGGTTTTCGCGTTAGAGAC-3′ and vimentin methylated antisense, 5′-CGACTAAAACTCGACCGACTCGCGA-3′. PCR amplification consisted of 40 cycles (95°C for 5 sec and 55°C for 30 sec) following an initial denaturation step (95°C for 10 sec). Genomic DNA, methylated in vitro by CpG methyltransferase (Sss I; New England BioLabs, Inc., Ipswich, MA, USA), served as a positive control and a water blank served as a negative control. GAPDH served as an internal control for normalization. The percentage of vimentin promoter methylation in each sample was estimated using the following formula: Methylated vimentin (%)=MM+U× 100%=11+2(−ΔCt)× 100%

Gene expression in gastric cell lines and tissue samples was quantified by RT-qPCR using a 7500 Real-Time PCR Platform (Applied Biosystems, Foster City, CA, USA). Vimentin and GAPDH expression levels were assessed using pre-designed TaqMAN probes VIM-Hs00185584_m1 and GAPDH-Hs02758991_g1, respectively (Applied Biosystems Life Technologies). Complementary DNA samples from three independent biological experiments were examined by RT-qPCR (50°C for 2 min, denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C annealing for 1 min). For each experiment, the samples were analyzed in duplicate. Normalized vimentin expression was determined using the comparative Ct (ΔΔCt) method using Relative Quantification Study software (7300 Sequence Detection system, version 1.4; Applied Biosystems Life Technologies).

To further examine whether vimentin expression is regulated by DNA promoter methylation, the AGS cell line, which demonstrated the highest level of vimentin gene methylation among the six cell lines, was selected for further study. The AGS cell line was cultured in RPMI-1640 medium supplemented with 10% FBS. Cells in the logarithmic proliferative phase were seeded into a 96-well plate (Corning, New York, NY, USA) at a density of 5,000 cells/well and cultured in an incubator (Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂ saturated humidity for 24 h. Cells were subsequently treated with 1 µM 5-Aza-dC (Sigma-Aldrich, St. Louis, CA, USA) for 72 h, with replenishment of fresh medium containing 5-Aza-dC every 24 h. Cells in the treatment and control groups were then harvested. Demethylation of the vimentin promoter regions was detected using MSP, and vimentin mRNA expression levels were measured using RT-qPCR. MSP and RT-qPCR were performed as described previously in the current study.

Statistical analyses were performed using SPSS (version 17.0; SPSS, Inc., Chicago, IL, USA). Vimentin DNA methylation was compared between diffuse-type and intestinal-type cell lines or between tumor and paratumor normal tissue samples by Student's t-test, and correlation of vimentin promoter methylation with vimentin expression was assessed by Pearson's correlation analysis. The χ² test or Student's t-test were utilized to compare the clinicopathological data. P<0.05 was considered to indicate a statistically significant difference.

Clinicopathological data were assessed for differences between diffuse- and intestinal-type GC cells. There were 36 (56.3%) patients with diffuse-type and 28 (43.7%) patients with intestinal-type GC. As shown in Table I, a significant difference was observed between diffuse- and intestinal-type GC for age (P=0.023), tumor (T) stage (P=0.020), node (N) stage (P=0.020) and Tumor-Node-Metastasis (TNM) stage (P=0.039). There was no significant difference observed for gender (P=0.072) or maximal tumor size (P=0.264).

In order to investigate epigenetic silencing mechanisms of the vimentin gene in GC, qMSP technology was applied to evaluate the DNA methylation status of the vimentin promoter. GES-1 immortalized human non-neoplastic gastric epithelial cells, intestinal-type GC cell lines (MKN-28, AGS, MKN-1) and diffuse-type GC cell lines (SGC-7901, SNU-5, KATO III), as well as tumor and paratumor normal tissue samples were analyzed. All GC cell lines and tissues that were assessed demonstrated promoter hypermethylation (>10%), and methylation levels were significantly higher in intestinal-type compared with diffuse-type (P<0.0001; Fig. 1A and B). Conversely, GES-1 and paratumor normal tissues exhibited hypomethylation at the vimentin promoter (P<0.0001; Fig. 1A and B).

Subsequently, RT-qPCR was conducted to investigate the correlation between the vimentin promoter methylation and expression levels. Vimentin expression levels were strongly detected in diffuse-type cell lines and tissues, weakly detected in intestinal-type cell lines and tissues, and markedly reduced in GES-1 and paratumor normal tissues. Through comparison of vimentin promoter methylation and vimentin mRNA expression levels, an inverse correlation was identified (Table II) in the GC cell lines (Fig. 2A and B) and tissues (Fig. 2C and D). This may suggest that expression is negatively regulated by vimentin promoter DNA methylation.

To evaluate the expression of vimentin in the various Lauren histological types, vimentin expression levels in intestinal-type cell lines and GC tissues were compared with those in diffuse-type cell lines and GC tissues. The results revealed significantly lower vimentin expression levels in intestinal-type cells and GC tissues when compared with diffuse-type cells and GC tissues (P<0.001; Fig. 3).

Among the six cell lines examined, AGS cells demonstrated the highest levels of vimentin gene methylation and thus, this cell line was selected for further study. To further examine whether vimentin expression was regulated by vimentin promoter DNA methylation, AGS cells were treated with a DNA demethylating agent (5-Aza-dC) for 72 h. Following treatment, promoter DNA methylation was significantly reduced, which was accompanied by reactivation of vimentin expression (P<0.01; Table III). Although significantly increased, vimentin expression was still in the range for GC.