The 9 GAC cell lines (MKN28, KATOIII, MKN45, SNU16, SNU1, MKN7, MKN1, NCI-N87 and AGS) (17), were cultured in RPMI-1640 (Gibco) supplemented with 10% fetal bovine serum (FBS; EU Gibco), 100 U/ml penicillin and 10 μg/ml streptomycin in a humidified atmosphere of 5% CO₂ at 37°C. Archival tissue blocks from 123 patients with diffuse-type GAC were retrieved from the Department of Anatomical and Cellular Pathology, Prince of Wales Hospital (PWH), Hong Kong. Frozen tissues from another 6 pairs of primary GACs and corresponding non-tumorous gastric mucosa were collected from patients who underwent gastectomy before any therapeutic intervention. The AJCC (7th edition) was used for cancer staging.

Total RNA was extracted using RNeasy Mini kit according to the manufacturer’s instruction (Qiagen). High-Capacity cDNA reverse transcription kits (Applied Biosystems) were applied to achieve cDNAs. For qRT-PCR, SYBR-Green PCR Master Mix (Applied Biosystems) was applied for MCM7 expression (sense, ACTT GTGGAATGGCAGGAAG and antisense, CTAGGCATCC CACTCAAAGG). The normal gastric mRNA was from Ambion (AM 7996). The relative expression level was normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and calculated using the 2^−ΔΔCt method. Quantitative PCR was performed in triplicate reaction wells including positive and negative controls.

Immunohistochemical staining of MCM7 was performed in 4 μm-thick sections. After de-waxing in xylene, sections were rehydrated, rinsed in distilled water, and incubated in 3% H₂O₂ solution for 25 min to block endogenous peroxidase activities. Antigen retrieval was done by using pressure cooker with 10 nM citrate buffer (pH 6.0) for a total of 30 min (high power 10 min followed by low power 20 min). The primary antibody MCM7 (1:1,000, CDC47 Ab-2; NeoMarkers, Fremont, CA, USA) was applied at 4°C overnight and chromogen development was performed using the standard avidin-biotin method (Dako). The nuclear expression of MCM7 was scored by estimating proportion of tumor cells with positive nuclear staining into 4 different groups (negative, none; weak, ≤10%; moderate, 10 to ≤25%; strong, >25%).

Protein was extracted from the GAC cell lines, normal gastric epithelium tissues together with 6 paired primary samples using RIPA lysis buffer with proteinase inhibitor. Protein mixed (20 μg) with 5X SDS loading buffer was loaded per lane, separated by 12% SDS-polyacrylamide gel electrophoresis. MCM7 protein was detected with MCM7 antibody (1:50, CDC47 Ab-2; NeoMarkers). Cleaved-PARP (Asp214) (1:1,000, #9541; Cell Signaling Technology, Danvers, MA, USA) was also evaluated to check late apoptosis.

Transfection was performed using Lipofectamine 2000 transfection reagent (Invitrogen) and siMCM7 (SI05064276 and SI05064283; Qiagen) at the concentration of 25 nM. Scramble siRNA was included as control. Cell proliferation was assessed using CellTiter 96^® Non-Radioactive Cell Proliferation Assay (MTT) (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The absorbance at 570 nm was measured and documented for the 5-day MTT proliferation analysis (Victor3; Perken-Elmer, Waltham, MA, USA). Monolayer colony formation assay was performed as described in our previous report (18). We used 6-well plates for this assay, 3 for siMCM7 and 3 for siScramble. The time-point was 8 days after transfection. Colonies were fixed with 70% ethanol for 15 min and stained with 2% crystal violet. Colonies with cell number of >50 cells/colony were counted. Cell invasion assay was performed in a 24-well invasion chamber (BD BioCoat Matrigel Invasion Chamber; BD Biosciences). Cells were harvested from the culture dishes 24 h after transfection, washed three times with culture medium containing 1% FBS, and resuspended in medium with 1% FBS. Then, 300 μl of the cell suspension (1×10⁵ cells) was added into the Transwells, with 500 μl of culture medium containing 10% FBS in the lower chamber. After 24-h incubation at 37°C, cells that invaded through the Matrigel membrane were fixed with 100% methanol for 2 min and stained with 1% Toluidine blue for another 2 min. Cells on the underside of the membrane were counted from 3 microscope fields and the average number recorded.

In the functional assays like MTT proliferation, monolayer colony formation and cell invasion, Student’s t-test was used to compare the phenotype difference of siMCM7 knockdown cells and siScramble transfected cells. Correlations between MCM7 expression with other clinicopathologic parameters were evaluated by non-parametric Spearman’s rho rank test. The Kaplan-Meier method was used to estimate the survival rates according to MCM7 nuclear staining. For the parameters statistically significant in the univariate survival analysis (P<0.05), the multivariate survival was achieved by the Cox regression analysis. All statistical analysis was performed by SPSS software (version 16.0; SPSS Inc.). A two-tailed P-value of <0.05 was considered to indicate a statistically significant result.

The MCM7 mRNA expression levels were higher in 6 GAC cell lines (KatoIII, MKN45, SNU16, MKN1, NCI-N87 and AGS) than it in the normal gastric tissue as shown in Fig. 1A. The upregulation of MCM7 in the GAC cells was further confirmed by western blot analysis. High MCM7 protein expression was observed in all GAC cell lines compared with the 3 normal gastric mucosal samples from patients who underwent weight reduction gastrectomy (Fig. 1B). Comparing tumor with the paired non-tumorous mucosa, upregulation of MCM7 protein was observed in all 6 tumor samples by western blot analysis (Fig. 1C). In normal gastric epithelium tissues, only cells from neck region (proliferation compartment) showed MCM7 nuclear expression by immunohistochemistry (Fig. 1D). Immunohistochemical analysis of 5 paired primary GAC samples showed positive nuclear staining of MCM7 in the tumor tissues but not in the non-tumorous gastric glandular epithelium (Fig. 1E).

Upregulation of MCM7 in GACs suggested a potential tumorigenic role of MCM7 in cancer development. To further elucidate the functional role it plays, we investigated the effect of MCM7 knockdown by siRNA in vitro. MCM7 were knocked down by 2 specific siMCM7s in AGS and NCI-N87 with high transfection efficiency (Fig. 2A). The 5-day MTT assay indicated that the MCM7 knockdown reduced cell proliferation rates to significant levels (AGS, 32.1 and 30.0%; NCI-N87, 40.5 and 43.6%; P<0.001) (Fig. 2B) compared with siScramble control groups. The viability change induced by siMCM7 was also confirmed by anchorage-dependent monolayer colony formation assays. A significant reduction of colony number and colony size was observed in cells transfected with siMCM7 compared with the siScramble group (reduced to 21.3 and 10.6% in AGS; 7.6 and 9.2% in NCI-N87; P<0.001) (Fig. 2C). In cell motility assays using BD BioCoat chamber, we confirmed that the invaded cell number was significantly lower in the siMCM7 transfected cells than the control groups (impaired to 23.0 and 19.1% in AGS; 34.9 and 47.6% in NCI-N87; P<0.001) (Fig. 2D), suggesting that MCM7 was involved in the invasion of cancer cells. Moreover, siMCM7 induced late apoptosis in AGS and NCI-N87 cells which was represented by activation of cleaved-PARP (Fig. 2A).

In total 267 GAC samples, MCM7 expression had no correlation with survival (P=0.724), thus, we checked its prognostic correlation according to histological type (intestinal and diffuse) and stage (early and advanced) separately. We found only in the diffuse-type GAC, MCM7 correlates with poor survival.

MCM7 was predominantly expressed in the nuclei of diffuse-type tumor cells (Fig. 3A). Of the 123 diffuse-type GAC samples, 16 cases (13.0%) had MCM7 negative nuclear staining, 11 cases (8.9%) had weak staining, 44 cases (35.8%) had moderate staining and 52 cases (42.3%) had strong staining. No significant survival difference was observed between the 4-grade MCM7 expression by univariate analysis (P=0.071; Fig. 3B). Then, we stratified negative and weak staining cases as negative/weak MCM7 expression group (27/123, 22.0%), and moderate and strong cases as moderate/strong MCM7 expression group (96/123, 78.0%). MCM7 nuclear expression correlates with poor survival based on these groupings (P=0.048; Fig. 3C).

We further investigated the clinicopathological correlation of MCM7 in diffuse-type GACs. Table I summarizes the correlation of MCM7 with clinicopathological parameters in diffuse-type GAC patients. MCM7 expression only correlated with advanced age (>60 years; P=0.015). Univariate analysis (Table II) indicated that MCM7 expression in diffuse-type GACs associated with poor disease-specific survival (P=0.048). Advanced age and stage were (T, N and M, respectively) also correlated with poor survival (P<0.05). By multivariate Cox proportional hazards regression analysis (Table II), only stage (T, N and M, respectively) was independently associated with disease-specific survival (P<0.05).