A total of 108 samples of tumor and adjacent tissue specimens from patients with gastric cancer who were diagnosed and received surgery at the Department of Surgery, Affiliated Hospital of Jiangnan University (Wuxi, China) were obtained between June 2012 and July 2014. None of the patients received preoperative chemotherapy or radiotherapy. Cases were excluded if they had been diagnosed with previous, recurrent or metastasized cancer from another origin. The samples were ground for protein extraction. The corresponding non-neoplasia mucosa tissues, which were resected ≥5 cm away from the tumor margin, served as controls. Clinicopathological data were obtained from a retrospectively constructed medical database, which had been reviewed and confirmed by two pathologists. The present study was approved by the Research Ethics Committee of Jiangnan University (approval no. JDFY20220801-1), and all patients provided written informed consent for the use of their data in research.

The adenoviral vectors pAd-LacZ (encoding β-galactosidase), pAd-IQGAP1 and pAd-Menin were kindly gifted by Dr Yong-Chang Chen (Jiangsu University, Zhenjiang, China). Mouse anti-menin antibody (EPR3986; product code ab92443) was obtained from Abcam, while mouse anti-IQGAP1 (cat. no. sc-376021), mouse anti-phosphorylated (p)-Akt (cat. no. sc-377556), mouse anti-Akt (cat. no. sc-5298), mouse anti-NF-κB (cat. no. sc-8414) and goat anti-β-actin antibodies (cat. no. sc-8432), as well as short-interfering RNAs (siRNAs) for IQGAP1 (5'-AAGTTCTACGGGAAGTAATTG-3') and menin (5'-CCACCUUUCUUGUGCAGUCCCUA-3'), and negative control siRNAs (RNA sequence, 5'-AUGAACGUGAAUUGCUCAA-3') were purchased from Santa Cruz Biotechnology, Inc. The horseradish peroxidase (HRP)-conjugated secondary antibody (cat. no. KCB002) was purchased from Rockland Immunochemicals Inc. Immunohistochemical and ECL reagents were acquired from EMD Millipore. All other reagents were of analytical grade.

The AGS cell line (cat. no. TCHu232) was purchased from the Institute of Cell Biology of the Chinese Academy of Sciences. The human gastric epithelial cell line GES-1 was a kind gift from Dr Yong-Chang Chen (Jiangsu University, Zhenjiang, China). The cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) with 10% new-born calf serum (NBCS; Lanzhou Minhai Bio-Engineering Co., Ltd.) in an incubator with 5% CO₂ at 37˚C. The culture medium was changed every 2 days, and the cells were sub-cultured until reaching confluence.

Adenovirus encoding menin or IQGAP1 gene at an MOI of 100 pfu/cell was used to infect human gastric cancer AGS cells. The expression level of target protein in AGS cells infected with the above recombinant adenovirus was detected by western blotting. For transfection, a complex of Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) and siRNA was prepared according to the manufacturer's instructions. Briefly, cells were seeded to 80% confluence and infected at 37˚C for 6 h using 100 pmol siRNA. After 6 h, the siRNA/lipid complexes were removed, and the cells were maintained in complete medium for an additional 48 h. The protein expression was then determined by western blotting.

The target proteins were extracted and detected by western blotting. Frozen tissue samples and cells were homogenized in RIPA buffer (Sigma-Aldrich; Merck KGAa). The homogenates were heated in boiling water for 5 min, and the proteins in the supernatant were quantified by Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc.). In total, 20 µg protein extracts were separated using 10% SDS PAGE and transferred onto a PVDF membrane. After blocking with 10% non-fat milk in TBS with 0.1% Tween-20 for 1 h at room temperature (RT), the membrane was incubated with specific antibodies against menin (1:500), IQGAP1 (1:1,000), p-Akt (1:1,000), Akt (1:1,000), NF-κB (1:1,000) and β-actin (1:1,000 as a loading control) overnight at 4˚C. Subsequently, the membrane was incubated with the corresponding HRP-conjugated secondary antibodies (1:1,000) for 1 h at room temperature, followed by three washes. The proteins bands were visualized using ECL. Images were analyzed using the imaging system (iBright™ CL750 Imaging System; Thermo Fisher Scientific Inc.).

AGS cells were infected with pAd-IQGAP1 or pAd-Menin to overexpress IQGAP1 or menin, respectively, whereas siRNAs were transfected into AGS cells to knock down the expression of menin or IQGAP1. The cells were serum-starved for 12 h and then trypsinized, washed, counted and re-suspended in 96-well plates (10,000 cells in 100 µl DMEM) for various time periods (12, 24, 48 and 72 h). Cell proliferation was measured using MTT assay. Briefly, 20 µl of MTT dye (5 mg/ml) was added to each well, and the plate was incubated for 4 h. Dimethylsulfoxide (DMSO; 150 µl) was added to the wells to dissolve the formazan crystals. The absorbance value at 450 nm was measured for each sample, and all the experiments were repeated three times with ≥3 replicates.

Data are expressed as the mean ± standard deviation. Unpaired two-tailed Student's t-test followed by Bonferroni's post hoc test were used to analyze differences among the variables. One-way ANOVA followed by Dunnett's post hoc test was employed to analyze differences between multiple sets of data. The association between each independent clinicopathological variable and menin or IQGAP1 was examined by χ² test. P<0.05 was considered to indicate a statistically significant difference.

Total proteins were extracted from 108 gastric cancer and paired normal tissues. Western blotting was used to detect the expression of menin and IQGAP1 proteins. According to the relative intensity of the protein bands (calculated as the ratio of the median of the gray scale bands of target proteins to β-actin), the samples were separated into an expression group (≥0.467 for menin and ≥0.57 for IQGAP1) and a no-expression group (<0.467 for menin and <0.57 for IQGAP1). The median of the gray scale bands of menin protein/β-actin was 0.315 in 108 gastric cancer tissues and 0.800 in paired normal tissues. The median of the gray scale bands of IQGAP1 protein/β-actin was 0.852 in 108 gastric cancer tissues and 0.371 in paired normal tissues (Fig. S1). As shown in Fig. 1, and Table I, menin expression was significantly lower in 23.1% (25 out of 108) gastric cancer tissues than in 76.9% (83 out of 108) paired non-neoplastic mucosa tissues (P<0.001). However, IQGAP1 was positively expressed in 66.7% (72 out of 108) gastric cancer tissues and 33.3% (36 out of 108) paired normal tissues (P<0.001). A negative association was identified between menin and IQGAP1 expression in gastric cancer tissues.

The protein expression of menin in AGS cells was significantly lower than that in GES-1 cells. However, the expression of IQGAP1 in AGS cells was significantly higher than that in GES-1 cells (Fig. 1B). The expression of menin and IQGAP1 was overexpressed in AGS cells infected with pAd-IQGAP1 and pAd-Menin, while the expression of IQGAP1 was knocked down in AGS cells transfected with IQGAP1 siRNAs (Fig. 2). The expression of menin and IQGAP1 in cells infected with pAd-IQGAP1 and pAd-Menin or transfected with IQGAP1 siRNAs for 12, 24, 48 and 72 h was also detected, and the expression of menin and IQGAP1 was observed to be increased or decreased in a time-dependent manner. The expression reached the highest or lowest level at 48 h post-infection, and remained at a stable level thereafter; thus, 48 h was used in subsequent experiments (Fig. 3). Of note, the expression levels of menin were not significantly altered over the period of time assessed in the menin siRNA groups compared with those in the control group. (Figs. 2 and 3).

The association between menin and IQGAP1 expression levels and clinicopathological parameters is summarized in Table II. The results showed that menin and IQGAP1 expression were associated with histological grade (P=0.047 and P=0.044, respectively), distant metastasis (P=0.033 and P=0.028, respectively), lymph node (LN) metastasis (P=0.006 and P=0.04, respectively) and TNM stage (P=0.014 and P=0.038, respectively). However, no significant associations were observed with patient sex (P=0.662 and P=0.561, respectively) or age (P=0.349 and P=0.395, respectively).

To determine whether the increase or decrease in one protein would affect the expression of the other protein, menin and IQGAP1 were overexpressed with pAd-Menin and pAd-IQGAP1, respectively. siRNAs targeting menin and IQGAP1 were transfected into AGS cells to knock down the expression of menin and IQGAP1. Western blotting was used to detect the expression of menin and IQGAP1. The results revealed that exogenous expression of menin repressed IQGAP1 expression. However, increased or knocked down expression of IQGAP1 did not affect the expression of menin. There was no change in menin or IQGAP1 expression levels in the menin siRNA group compared with those in the control group (Fig. 2).

The effects of menin and IQGAP1 on cell proliferation were evaluated by MTT assay. AGS cells were transfected with siRNAs, which interfered with the expression of menin and IQGAP1. In addition, AGS cells were infected with an adenovirus vector encoding IQGAP1 or menin for overexpression. The results revealed that ectopic expression of IQGAP1 promoted, whereas silencing of IQGAP1 expression inhibited, gastric cancer cell proliferation. On the other hand, increased expression of menin suppressed cell proliferation. Because menin protein is less expressed in AGS cells, menin siRNA could further reduce menin expression in AGS cells (Fig. 3). The results revealed that menin suppressed, whereas IQGAP1 stimulated, the proliferation of gastric cancer cells.

To explore the potential mechanism by which menin suppressed the proliferation of gastric cancer cells, the effects of menin on the expression of crucial proteins of the PI3K and NF-κB signaling pathways were examined. The levels of p-Akt and NF-κB were notably downregulated in menin-overexpressing AGS cells, while no changes in Akt were observed (Fig. 4).