The BGC823 GC cell line was purchased from the Xiangya School of Medicine Central Experiment Laboratory (Changsha, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS; Gibco Life Technologies), 100 U/ml penicillin and 0.1 mg/ml streptomycin, at 37°C in a 5% CO₂ humidified atmosphere.

The primary antibodies against IGF-1R and β-actin, were purchased from Abcam (Cambridge, UK), and Sigma-Aldrich (St Louis, MO, USA), respectively. Thegoat anti-mouse Immunoglobulin G (IgG) and goat anti-rabbit IgG secondary antibodies were purchased from Kirkegaard & Perry Laboratories, Inc. (Gaithesburg, MD, USA).

BGC823 cell suspensions were seeded (4×10⁴ cells/well) in a 96 well plate. Following a 16 h culture, the cells were infected with pSUPER-IGF-IR-siRNA2 or pSUPER-control-siRNA2 (Genechem, Shanghai, China), or treated with phosphate-buffered saline (PBS) for 0, 24, 48 or 72 h. The cells were supplemented with 0.5 μg/μl MTT and cultured for a further 4 h, followed by treatment with dimethyl sulfoxide for 10 min. The absorbance of the samples was measured at 570 nm using an iMark platereader (Bio-Rad Laboratories, Hercules, CA, USA).

The BGC823 cells infected with pSUPER-IGF-IR-siRNA2 or pSUPER-control-siRNA2, or treated with PBS, were fixed with 70% ice-cold ethanol at 4°C overnight. The cells were then treated with RNase A (Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 30 min and propidium iodide (PI; Sigma-Aldrich) at 4°C for 1 h. The DNA content of the cells was assessed using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and the data were analyzed using Modfit 3.0 DNA software (BD Biosciences, Franklin Lakes, NJ, USA).

The BGC823 cells treated with the indicated siRNAs, or PBS, were incubated with 20 μg/ml RNase A and stained with 50 μg/ml PI solution. A total of 500 cells were randomly counted under a microscope (Axio observer Z1; Zeiss Meditec, Jena, Germany). The apoptotic rate was calculated using the following formula: Rate of cell death = stained cells/total cells.

The cells infected with the siRNAs, or treated with PBS, were treated with Tris-NaCl-EDTA buffer, containing proteinase K, at 55°C overnight. Genomic DNA was isolated from the cells using DNA purification kits (Life Technologies, Carlsbad, CA, USA) and fractionated by 2% agarose gel electrophoresis.

The cells were infected with the specified siRNAs, or treated with PBS for 48 h, prior to being seeded in a fibronectin-coated 6 well plate and cultured until confluent. The cells were then cultured in serum-free DMEM and a straight scratch was made using a 200 μl pipette tip. The width of the scratch was observed and recorded using a microscope, at 0, 6, 12, 24 and 36 h. Cells of three independent wound areas were counted. A minimum of three wells were counted per experiment.

Transwell chambers were sterilized by UV treatment, and the inserts and PVDF membrane were coated with Matrigel^® matrix (Life Technologies). The cells (1×10⁵), in serum-free media, were plated in the upper wells of the chambers, and media supplemented with 10% FBS was added to the lower wells. Following a 24 h incubation, the cells on the Matrigel^® side of the chambers were removed using a cotton swab. The inserts were fixed in methanol and stained using 0.2% crystal violet staining solution (Life Technologies).

The data are expressed as the means ± standard deviation. Statistical differences were evaluated using an unpaired student’s t test using SPSS version 15.0 (SPSS Inc., Chicago, IL, USA) software. A value of P<0.05 was considered to indicate a statistically significant difference.

An MTT assay was performed to determine whether silencing of IGF-1R expression influenced gastric cancer cell growth. The cells were infected with pSUPER-IGF-IR-siRNA2 or pSUPER-control-siRNA2, or treated with PBS, for 0, 24, 48 and 72 h. Knockdown of IGF-1R expression for 24 h did not affect cell growth, as compared with the control siRNA and PBS groups. However, IGF-1R ablation for 48 to 72 h, significantly reduced cell growth by 62.15 and 59.82%, respectively (Table I and Fig. 1). This observation indicates that the greatest effects of IGF-1R expression knockdown on cell growth inhibition were achieved 48 h post-infection.

To determine whether silencing of IGF-1R expression affected the progression of the cell cycle a flow cytometric analysis was performed. The cells were fixed with ethanol 48 h post-infection, stained with PI and subjected to fluorescence-activated cell sorting. The percentages of the IGF-1R-depleted cells in the G₀/G₁, S and G₂/M phases were 59.42, 37.83 and 3.71%, respectively (Table II and Fig. 2). The control cells, either transfected with control siRNA or treated with PBS, had a significantly lower G₀/G₁ cell population and higher S population (Table II and Fig. 2). These data demonstrate that knockdown of IGF-1R expression results in G₁ cell cycle arrest, and a subsequent reduction in the S phase population, in gastric cancer cells.

An accumulated sub-G₁ population was observed in response to IGF-1R expression knockdown, suggesting an increased number of apoptotic cells (Fig. 2). To further confirm whether IGF-1R ablation triggered apoptosis in gastric cancer cells, PI staining and microscopic analysis were performed. Living cells with an intact cytomembrane are resistant to PI staining; however, apoptotic cells are easily stained. The percentages of living cells in the IGF-1R siRNA, control siRNA and PBS groups were 8.43, 49.72 and 47.53%, respectively (Fig. 3). These results indicate that IGF-1R ablation was capable of inducing apoptosis. A DNA laddering analysis was also conducted and IGF-1R expression knockdown dramatically enhanced the formation of a DNA ladder, which could barely be seen in the control cells (Fig. 3D). These results demonstrate that silencing of IGF-1R expression induces gastric cancer cell apoptosis.

Wound-healing assays were conducted to assess the migratory ability of BGC823 cells in response to IGF-1R ablation. IGF-1R ablation markedly suppressed cell motility at 36 h post-infection. The control siRNA or PBS treated cells migrated to heal the wound areas, whereas the IGF-1R-depleted cells were incapable of migrating (Fig. 4A–C). Furthermore, the cells of the wound areas were counted, and the number of cells were significantly lower in the IGF-1R depletion group, as compared with the control groups (Fig. 4D). These results indicate that suppression of IGF-1R expression inhibits gastric cancer cell motility.

An in vitro Transwell assay was used to determine whether IGF-1R ablation would affect gastric cancer cell invasion (Fig. 5A–C). The average number of invasive IGF-1R-depleted cells was 98.33 (Fig. 5D), which was significantly reduced, as compared with the control siRNA (128.33) and PBS groups (135.33) (Fig. 5D). These results suggest that IGF-1R depletion may also prevent gastric cancer cell invasion.