A total of 66 tissue specimens were collected from the Third Xiangya Hospital (Changsha, China) in the present study, including 47 gastric cancer tissue specimens (21 female and 26 male) and 19 normal gastric tissue specimens (8 female and 11 male). The median age of the patients was 48.67 years (range, 26–84 years), and the samples were collected between September 2013 and September 2015. The specimens were stored at −80°C and used for western blot analysis. The human investigations in the present study were performed in compliance with the Declaration of Helsinki. Approval for the study was obtained from the Ethics Committee of Third Xiangya Hospital. The patients were well informed of the details and signed relevant consent forms prior to commencement of the investigation The experiments using mice in the present study were performed in accordance with Animal Ethical Care (24,25).

The BGC-823 and MKN-45 gastric cancer cell lines were obtained from Shanghai Institute of Materia Medica, Chinese Academy of Tumor Cell Bank (Shanghai, China). The cells were cultured at 37°C and 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (GE Healthcare Life Sciences, Logan, UT, USA), 1% penicillin and 1% streptomycin. Skp2 inhibitor C1 (SKPin C1) was purchased from MedChem Express (Princeton, NJ, USA). A purified recombinant protein of human p45 Skp2, transcript variant 1 with an N-terminal His tag, expressed in Escherichia coli (50 µg), was obtained from OriGene Technologies (Beijing, China). MTT reagent was obtained from Sigma-Aldrich (St. Louis, MO, USA). The human Skp2 short hairpin (sh)RNA, four unique 29-mer shRNA constructs in the retroviral green fluorescent protein (GFP) vector, and the non-effective 29-mer scrambled shRNA cassette in the pGFP-V-RS vector were obtained from OriGene Technologies. Bioinformatics analysis was conducted using the BioGPS gene annotation portal (http://biogps.org/).

The cells were lysed with radioimmunoprecipitation assay lysis buffer (P0013B), which was obtained from Beyotime Institute of Biotechnology (Haimen, China). The proteins in the lysates were separated by 10% polyacrylamide gel electrophoresis, which was prepared in house including a stacking gel and a separating gel. The proteins were loaded on a polyvinylidene difluoride membrane. The antibodies used in the experiments were as follows: Rabbit polyclonal anti-Skp2 antibody (1:1,000; cat. no. 15010-1-AP) was obtained from ProteinTech Group, Inc. (Wuhan, China). Rabbit polyclonal anti-p27^kip1 antibody (1:1,000; cat. no. ab137736) was purchased from Abcam (Cambridge, MA, USA). Rabbit polyclonal anti-β-actin antibody (1:1,000; cat. no. sc-7210) and horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (sc-2030) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The blots were visualized using the GelDoc XR system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and Quantity One 1-D analysis software, version 4.6.9 (Bio-Rad Laboratories, Inc.).

The cell survival rate and proliferation were determined using an MTT assay, as described (26,27). In brief, the BGC-823 cells and MKN-45 cells were plated into 48-well plates at 1,000 cells/well, and the cells were transfected with Skp2-specific shRNA or negative control (N.C.) shRNA using Lipofectamine 2000 for 24, 48, 72 and 96 h, respectively, at room temperature. At 4 h prior to analysis, 5 mg/ml of MTT was added into the medium. Finally, the purple crystals was dissolved with DMSO, and the data were analyzed at a test wavelength of 490 nm.

For Skp2 inhibition, the human gastric cancer cells were plated into 96-well plates at 1,000 cells/well at 37°C with 5% CO₂. After 6 h, the BGC-823 cells and MKN-45 cells were treated with SKPin C1 at concentrations of 1, 5 and 10 µM for 48 h, and with 5 µM SKPin C1 for 24, 48 and 72 h. The other steps were as described above.

A total of 30 (10/group) C57BL/6 male nude mice (6–8 week-old; 18–20 g) were obtained from the Si Lai Ke Jing Da Laboratory Animal Co., Ltd. (Changsha, China). The mice were randomly divided into three groups and maintained in specific pathogen-free conditions at 22°C under a 12/12 h light/dark cycle with free access to food and water. The mice were challenged subcutaneously in the flank area with 5×10⁵ BGC-823 tumor cells, BGC-823 cells transfected with Skp2 shRNA or BGC-823 cells transfected with N.C. shRNA. The tumor volumes were recorded every 3 days. At 30 days post-tumor injection, the mice were sacrificed by cervical dislocation and the tumor were weighed. Each group contained >10 mice.

The data in the present study were analyzed using SPSS software (SPSS, Inc., Chicago, IL, USA). Student's t-tests were used to evaluate statistical significance. Data are expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

In order to determine the association between the expression of Skp2 and the progression of human gastric cancer, the presents study used bioinformatics methods to analyze the expression of Skp2 in the gastric tissues. BioGPS is a centralized gene portal for aggregating distributed gene annotation resources (28). The expression levels of Skp2 in the normal tissues, primary gastric cancer tissues and gastric cancer cell lines were readily and directly compared with each other. As shown in Figs. 1 and 2, the mean relative expression of Skp2 in the normal gastric tissues was 3.50, however, the mean values were 772 and 2,001.8 in the 31 primary gastric tumor tissues from the UK patient cohort and 10 gastric cancer cell lines of the side population, respectively. These data demonstrated that Skp2 was significantly upregulated in the primary gastric cancer tissues and gastric cancer lines, and that Skp2 may be an oncogene in the progression of gastric cancer.

In order to confirm the results of the bioinformatics analysis, tissue specimens from patients with gastric cancer and the specimens of normal gastric tissues were collected. Western blot analysis was used to determine the expression levels of Skp2 and p27^kip1, as degradation of the tumor suppressor, p27^kip1, is mediated by Skp2. In the present study, the data were normalized to the level of β-actin, followed by analysis using Student's t-test and presentation of data as the mean ± standard deviation. As shown in Fig. 3, three independent samples were used to detect the expression levels of Skp2 and p27^kip1, and the results demonstrated that the expression levels of Skp2 in the gastric cancer tissues were significantly increased, compared with those in the normal gastric tissues. However, the expression levels of p27^kip1 were inversely correlated with the levels of Skp2.

As shown in Table I, the expression levels of Skp2 in tissue specimens from 47 patients with gastric cancer and 19 normal gastric tissue specimens were assessed and analyzed using western blot analysis. Positive expression of Skp2 was observed in 41 specimens (87.2%) of the gastric cancer samples, whereas the positive rate of expression was 5.6% in the normal gastric tissue samples (P<0.01). The results were consistent with those of the bioinformatics analysis.

In order to determine whether inhibiting the expression of Skp2 affects the proliferation of human gastric cancer cells, the present study used Skp2-specific shRNA to interfere with the endogenous expression of Skp2. As shown in Fig. 4A, the human gastric cancer cells were transfected with Skp2 shRNA and cultured for 24, 48, 72 and 96 h, respectively. The subsequent MTT assay showed that the Skp2 shRNA-transfected cells had significantly lower survival rates, compared with the cells transfected with the N.C. shRNA. By contrast, the administration of recombinant Skp2 protein at a concentration of 0.1 µg/ml promoted the proliferation of the BGC-823 cells and MKN-45 cells, although no statistically significant difference was found between the recombinant Skp2-treated cells and the untreated gastric cancer cells.

In order to confirm the role of Skp2 in the proliferation of human gastric cancer cells, an Skp2 inhibitor was to treat the BGC-823 cells and MKN-45 cells. SKPin C1 is a potent inhibitor of Skp2, and selectively inhibits Skp2-mediated p27^kip1 degradation by targeting the SCF-Skp2 protein-protein interface. As shown in Fig. 4B, the BGC-823 cells and MKN-45 cells were treated with increasing concentrations of SKPin C1 for 48 h. The subsequent MTT assay results demonstrated that, as the concentrations of the Skp2 inhibitor increased, the survival rate of the human gastric cancer cells decreased significantly, compared with that of the untreated cells.

To examine the effects of Skp2 over time, 5 µM was selected as an appropriate middle-range concentration for treatment of the human gastric cancer cells. As shown in Fig. 4C, the BGC-823 and MKN-45 cells were treated with 5 µM of SKPin C1 for 24, 48 and 72 h, respectively. The results revealed that the proliferation of the BGC-823 cells and MKN-45 cells were significantly suppressed, compared with the untreated cells, in a time-dependent manner.

The present study also investigated the association between the levels of Skp2 and p27^kip1 in human gastric cancer cells. RNA interference technology and the specific inhibitor of Skp2 were used to interrupt the effect of Skp2 in the BGC-823 cells. As shown in Fig. 5A, the BGC-823 cells were transfected with Skp2 shRNA, to interfere with the expression of endogenous Skp2, for 48 and 72 h. The Skp2-depleted BGC-823 cells showed marked accumulation of p27^kip1, compared with the N.C. shRNA-transfected BGC-823 cells. Consistent with the RNA interference experiment, the present study used increasing concentrations of Skp2 inhibitors to treat the BGC-823 cells for 48 h, The results revealed that the endogenous expression of Skp2 was not affected significantly by SKPin C1, however, the accumulation of p27^kip1 in the BGC-823 cells was significantly upregulated as the concentration of SKPin C1 increased. These data suggested that the Skp2-mediated degradation of p27^kip1 was inhibited by Skp2 shRNA transfection and Skp2 inhibition.

To further identify the inhibitory efficacy of Skp2 deletion in human gastric cancer cells, tumorigenicity experiments were performed in nude mice. The nude mice were randomly divided into three groups (>10 mice per group), as follows: Skp2 shRNA group, N.C. shRNA group and untreated group. The stably transfected BGC-823 cells were subcutaneously injected into the mice. As shown in Fig. 6, the tumor weight in the Skp2 shRNA group was significantly lower, compared with that in the N.C. shRNA group (P<0.01) and untreated group (P<0.01). This was consistent with the results obtained in the analysis of tumor volume in the tumorigenicity experiments (P<0.01, vs. N.C. shRNA group).