Forty-eight specimens of GC tissues and adjacent non-cancer tissues were surgically obtained between June 2012 and December 2013 at the First Affiliated Hospital of Nanjing Medical University (Jiangsu, China). The corresponding normal gastric tissue samples were extracted >5 cm from the edge of the tumor, while no evident organization of the tumor cells was detectable. Resected tissue samples were immediately frozen in liquid nitrogen, and stored at −80°C until the extraction of total RNA. TNM disease stage was classified according to The American Joint Committee on Cancer (AJCC), 7th edition. In the present study, patients that had undergone any preoperative treatments were not included.

The GES-1 human gastric epithelial mucosa cell line, and SGC7901, MKN28, MKN45, AGS and BGC823 GC cell lines were obtained in our general laboratory. The cells were cultured in RPMI-1640 containing 10% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 μg/ml) under cell culture conditions (5% CO₂, 95% relative humidity and 37°C).

Total RNA was isolated from cell cultures or tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). First-strand complementary DNA (cDNA) was reverse-transcribed using the PrimeScript RT kit (Takara, Dalian, China). RT-qPCR was performed with FastStart Universal SYBR-Green Master (Rox) (Roche Diagnostics, Indianapolis, IN, USA) with an ABI 7500 (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA, USA). The specific oligonucleotide primers are listed in Table I. PCR was performed using the following cycles: 95°C for 30 sec, 40 cycles of 95°C for 5 sec, 60°C for 30 sec; and the dissociation stage: 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec. Each sample was analyzed in triplicate.

To investigate the overexpression of IFITM3, we modified LV-IFITM3 vector lentiviral constructs (Shanghai GenePharma Co., Ltd., Shanghai, China) to knock down IFITM3 in GC cells. The empty lentiviral (LV-NC) with enhanced green fluorescent protein (EGFP) served as a negative control. The constructed vectors were verified by DNA sequencing. The GC cells were infected with LV-IFITM3 and LV-NC. The supernatant was removed after 24 h and replaced with fresh culture medium. The cells were selected with 2 μg/ml puromycin for 2 weeks.

Cells (5×10⁵) were seeded in 6-well plate and incubated for 24 h. When the cells were grown to 90–100% confluency, a linear wound was generated by scratching the adherent cells using a sterile plastic pipette tip. The medium was displaced with serum-free medium. Immediately and 24 h after incubation at 37°C, migrating cells at the wound front were photographed using an inverted microscope. Each experiment was performed at least three times in triplicate.

In vitro invasion was determined using 24-well Transwell chamber. Cells (2×10⁴) were suspended in 100 μl of serum-free medium and placed in the upper compartment of a Transwell chamber with 8-mm membrane filter inserts coated with Matrigel (BD Biosciences). Subsequently, the medium with 10% FBS was added to the lower chamber as a chemoattractant. After incubation for 24 h, the cells on the upper surface of the membrane filter were removed, and the cells that had penetrated to the lower surface of the membrane were fixed and stained with crystal violet. Five visual fields of each insert were randomly selected and counted under a light microscope. Migration assays were performed using a Transwell compartment, and with the exception of Matrigel, all other steps were identical. For each experimental condition, the assay was performed at least in triplicate.

Cell proliferation was measured by the CCK-8 (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Cells were seeded in a 96-well plate at 5×10³ cells/well with 100 μl of complete culture medium. After adhesion for 24 h, 10 μl CCK-8 was added to each well and incubated at 37°C for 2 h. The absorbance was measured at 450 nm using a microplate reader. Experiments were performed in triplicate and repeated three times.

Cells (3×10⁵ cells/well) were seeded in 6-well plates and cultured for 24 h. After adhesion, the cells were harvested and centrifuged at 1,500 rpm for 5 min. Pellets were washed with cold phosphate-buffered saline (PBS) and fixed with cold 70% ethanol at 4°C for 30 min and stored at −20°C overnight. The following day, the cells were centrifuged at 1,500 rpm for 5 min to remove ethanol and washed with PBS. The supernatant was discarded and propidium iodide (PI) containing RNase A was added at 4°C for 30 min in the dark. Cell suspension was filtered through a mesh filter and analyzed by flow cytometry.

Cultured cells were lysed using RIPA buffer (Beyotime Institute of Biotechnology), and equivalent amounts of protein were subjected to SDS-PAGE separation and subsequently diverted to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat milk in Tris-buffered saline solution containing 0.05% Tween-20 and then incubated with antibody-specific IFITM3 (1:1,000; ab109429, Abcam), E-cadherin, vimentin, β-catenin, MMP-2, MMP-9 (1:1,000), GAPDH (1:10,000) (both from Cell Signaling Technology). The membranes were washed three times with TBST, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:1,000; Beijing Biosynthesis Biotechnology) at 37°C for 2 h. Bound proteins were visualized using ECL (Pierce) after three TBST washes and detected using a Bio-Imaging System. GAPDH was used as an internal loading control.

For immunohistochemistry, the tissue samples were fixed in 10% formalin solution and embedded in paraffin. Previous sections of 3 mm were prepared, then deparaffinized in xylene, and rehydrated through a graded series of ethanol. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide in methanol for 20 min at room temperature. For pretreatment, antigen retrieval sections were performed in citrate buffer (pH 6.0) using a microwave for 10 min. Overnight incubation at 4°C was carried out for the binding of primary antibodies (IFITM3, 1:400; ab109429, Abcam). The secondary anti-mouse antibody was subsequently applied to the sections for 30 min at room temperature. After rinsing with TBST, the slides were treated with diaminobenzidine solution and counterstained with hematoxylin. Immunostaining intensity (0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining) as well as the percentage of cells stained (0, no cells; 1, <10% of cells; 2, 11–50% of cells; 3, 51–80% of cells; and 4, >80% of cells) were evaluated.

The Statistical Program for Social Sciences (SPSS) 20.0 software (IBM, SPSS, Inc., Chicago, IL, USA) was used for the statistical analysis. The data were expressed as means ± standard deviation (SD). Differences were analyzed with the unpaired Student’s t-test and analysis of variance (ANOVA). A two-tailed value of P<0.05 was considered to indicate a statistically significant result.

To examine the role of IFITM3 in GC progression, the mRNA and protein expression of IFITM3 in the AGS, SGC7901, BGC823, MKN45 and MKN28 human GC cell lines and the GES-1 human gastric epithelial mucosa cell line were analyzed by RT-qPCR and western blot analysis, respectively. As shown in Fig. 1A, the expression levels of IFITM3 mRNA and protein were significantly higher in SGC7901 and BGC823 cell lines than in normal cell lines. However, there was no significant difference for MKN28 and MKN45. Thus, SGC7901 and BGC823 were selected as the optimal experimental groups. Furthermore, we examined the expression in 48 pairs of GC tissues compared with adjacent normal tissues. IFITM3 levels were markedly upregulated in cancer tissues compared with corresponding non-cancer tissues (Fig. 1B). The potential related factors affecting IFITM3 expression were determined and it was confirmed that IFITM3 expression was significantly associated with tumor differentiation, lymph node and distant metastasis, and TNM stages. Nevertheless, there was no significant correlation between IFITM3 expression and other clinicopathologic characteristics, such as age, gender, diameter, tumor location and CEA values (Table II). We also examined the expression of IFITM3 by immunohistochemistry and it was found that IFITM3 was positively stained in the cytoplasm of the majority of invasive cancer specimens, whereas IFITM3 was negatively or weakly stained in the adjacent normal tissues. Furthermore, IFITM3 expression levels were increased with the pathological stages of GC (Fig. 1C).

To demonstrate the functional role of IFITM3 in GC tumorigenesis, we infected IFITM3 shRNA into SGC7901 and BGC823 cells and examined the transfection efficiency by RT-qPCR and western blot analysis, respectively. We found that the levels of IFITM3 expression in the LV-NC were markedly higher than that of LV-IFITM3 (Fig. 2A and B). Furthermore, obvious inhibitory effects on cell proliferation were observed in IFITM3-silenced cells at 4–6 days (Fig. 2C and D). These results suggested that IFITM3 overexpression contributes to the proliferation of colon cancer cells.

To elucidate the effect of IFITM3 downregulation on cell cycle distribution, the flow cytometry instrument analysis was performed to show the increasing proportion of G0/G1 phase in GC cells. As shown in Fig. 3A and B, there was a significant increase in the cell population at the G0/G1 phase, associated with a decrease in the cell population at the G2/M and S phases following IFITM3 depletion (P<0.05; Fig. 3C and D). These data suggested that depletion of IFITM3 arrests GC cells in the G0/G1 phase.

To exhibit the involvement of IFITM3 in the invasion of gastric cells, wound-healing and Transwell assays were performed using SGC7901 and BGC823 cells transfected with LV-IFITM1 or LV-NC. The cell migration effect identified by the wound-healing assay showed that compared with negative control vector-transfected cells, IFITM3 knockdown significantly inhibits GC cell migration (Fig. 4A). Similar results were obtained from the Transwell assay, while transfection with LV-IFITM3 significantly suppressed the migration and invasion of SGC7901 and BGC823 cells (P<0.05; Fig. 4B and C). EMT, an essential cell biological program during embryonic development, contributes to cancer invasion and metastasis (19,20). To confirm the role of IFITM3 in the process of GC cells, we transfected the LV-IFITM3 vector and evaluated the expression of epithelial marker E-cadherin and mesenchymal marker vimentin at mRNA and protein levels. As shown in Fig. 4D, IFITM3 knockdown cells induced round spheroids with no or few protrusions. The increased expression of E-cadherin and decreased expression of vimentin was detected in IFITM3 knockdown cells in the mRNA and protein levels (Fig. 4E and F). This suggested that IFITM3 may regulate the migration and invasion potential of the gastric cell lines by inducing EMT.

MMPs are matrix metalloproteinases that were previously shown to play a crucial role in the tumor microenvironment by enhancing cancer cell invasion, proliferation and cancer matastasis (21,22). The results showed that IFITM3 is able to promote the migration and invasion of GC cancer cells. However, whether IFITM3 mediates MMP expression remains to be determined. Therefore, we examined MMP-2 and MMP-9 expression in transfected cells and negative control by qPCR and western blotting, respectively. A significant downregulation of MMP-2 and MMP-9 expression was observed in IFITM3-silencing cells, as compared with the empty lentiviral cells (P<0.05; Fig. 5A and B). These results suggested that MMPs are involved in IFITM3-induced cell migration and invasion.

The canonical Wnt/β-catenin pathway plays an important role in embryonic development and tumorigenesis. It has been shown that Wnt/β-catenin signaling participates in the regulation of IFITM genes during intestinal tumorigenesis (23). Wnt signaling and stable axin protein leading to β-catenin Therefore, we investigated whether the upregulation of IFITM3 destruction (24). Concentrations of 5 and 10 μmol/l, and an in GC cells was induced by activation of Wnt/β-catenin optimal processing time of 48 h were selected. GC cells were signaling. XAV939, a new molecular inhibitor, blocked subsequently treated with various concentrations of XAV939 and IFITM3 expression was analyzed using RT-qPCR and western blot analysis. The results showed that the expression of β-catenin and IFITM3 maintained a high level in normal GC cells. After incubating with XAV939 with 5 and 10 μ/mol, IFITM3 expression markedly decreased together with the reduction of β-catenin, both of which were significantly inferior to the untreated controls (P<0.05; Fig. 6A and B).