The present study was approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University (Nanchang, China). Informed consent was provided by patients from whom tissue samples were obtained for investigation.

The human MGC803 and SGC7901 GC cell lines were purchased from the Type Culture Collection of the Chinese Academy of Science (Shanghai, China). GES-1, a normal human gastric mucosa cell line, and the AGS, BGC823 and HGC-27 GC cell lines were obtained from the Sun Yat-Sen University Cancer Center (Guangzhou, China). All cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences) for 24 h in a humidified chamber at 37°C and 5% CO₂.

A total of 4 fresh GC tissues and paired noncancerous gastric mucosal tissues were collected by gastrectomy from the Department of General Surgery of the First Affiliated Hospital of Nanchang University. All the fresh samples were immediately snap-frozen in liquid and stored at −80°. Paraffin-embedded GC tissues (n=90) and gastric normal tissues (n=60) were collected from the Department of Pathology of the First Affiliated Hospital of Nanchang University between 2007 and 2009. None of these 90 patients with GC had received neoadjuvant chemotherapy or radiation therapy prior to surgery. The overall survival (OS) of the patients with GC was defined as the period between the date of surgery to the time at which the patient succumbed to mortality from any cause, or the date of the last follow-up if no event was documented. Tumor stage was determined according to the 2010 American Joint Committee on Cancer criteria (20). Histological differentiation was based on World Health Organization criteria (21). The detailed clinical information of the patients is summarized in Table I.

The tissue samples were formalin-fixed overnight and then paraffin-embedded at room temperature, prior to being cut into 4-µm serial sections. The sections were deparaffinized and rehydrated by incubation in 3% hydrogen peroxide for 15 min at room temperature. Antigen retrieval was performed by heating the sections in citrate buffer at 95°C for 1 h. The sections were then incubated with rabbit anti-human polyclonal TRIM24 antibody (1:200 dilution; catalog no., ab70560; Abcam, Cambridge, UK) or rabbit anti-human monoclonal β-catenin antibody (1:100 dilution; catalog no. 8480; Cell Signaling Technology, Inc., Danvers, MA, USA) in a humidified chamber overnight at 4°C. Following washing three times with phosphate-buffered saline (PBS), the sections were incubated at room temperature with a horseradish peroxidase system and DAB substrate (Dako, Carpinteria, CA, USA), followed by incubation with PBS containing diaminobenzidine for 10 min at room temperature.

The tissue samples were examined by two investigators who were blinded to the pathological information, and immunoreactivity was scored using the German Semi-Quantitative method (22). For TRIM24 staining, each specimen was scored according to its staining intensity (0, none; 1, weak; 2, moderate; 3, strong) and the percentage of stained cells (0, 0%; 1, 1–24%; 2, 25–49%; 3, 50–74%; 4, 75–100%). A final staining index (SI) was calculated as the product of the intensity and percentage scores, yielding a value between 0 and 12, which was classified as negative (−, 0–1), weak (+; 2–4), moderate (++; 6–8) or strong (+++; 9–12). An SI ≥6 was considered a high expression of TRIM24 and an SI<6 was considered a low expression of TRIM24. For β-catenin, membrane or cytoplasmic staining was considered negative, whereas nuclear expression was considered positive. The scoring of TRIM24 and β-catenin were evaluated independently by two board-certified clinical pathologists blinded to the clinical parameters. Any discrepancy between the scores assigned by the two investigators was resolved by re-evaluation and careful discussion until a consensus was reached.

Total RNA was extracted with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and RNA concentration was measured using spectrophotometry. cDNA was obtained using an EasyScript First-Strand cDNA Synthesis Supermix kit (TransGen Biotech, Inc., Beijing, China) and RT-qPCR analysis was performed on an ABI Prism 7500 Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using SYBR Green qPCR Supermix-UDG with ROX (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The primers used for RT-qPCR analysis are listed in Table II. The target gene expression levels were was normalized against that of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase, and calculated as 2^{−[(Cq target gene) - (Cq GAPDH)]}, where Cq is the quantification cycle for each transcript (23).

The cells and tissues samples were lysed in radioimmunoprecipitaion assay buffer (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) supplemented with protease in inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). The protein concentration was determined by Bradford method with bovine serum albumin (BSA) as the standard. Equal quantities of protein (50 µg) from each group were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. Following incubation in blocking buffer for 1 h at room temperature, the membrane was incubated overnight at 4°C with the following primary antibodies: Rabbit anti-human polyclonal TRIM24 (1:1,000 dilution; catalog no. ab70560; Abcam), rabbit anti-human monoclonal β-catenin (1:1,000 dilution; catalog no. 8480; Cell Signaling Technology, Inc.), rabbit anti-human monoclonal c-Myc (1:1,000 dilution; catalog no. 5605; Cell Signaling Technology, Inc.), rabbit anti-human monoclonal cyclinD1 (1:1,000 dilution; catalog no. 2978; Cell Signaling Technology, Inc.) and mouse anti-human monoclonal β-actin (1:2,000; catalog no. 8H10D10; Cell Signaling Technology, Inc.). Following three washes in Tris-buffered saline with 0.1% Tween 20, the membrane was incubated with horseradish peroxidase-labeled anti-rabbit or anti-mouse secondary antibody (1:3,000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at room temperature and developed using a chemiluminescence detection system (Thermo Fisher Scientific, Inc.).

Small interfering RNAs (siRNAs) targeting TRIM24 and negative control (NC) siRNA were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The TRIM24 siRNA sequence was 5′-GCUGGACUCUCUAAACAAUTT-3′. In vitro transient transfection was performed using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The cells were divided into three groups: Blank, transfected with NC siRNA or transfected with TRIM24 siRNA, and grown to 70–80% confluency prior to transfection. TRIM24-knockdown was confirmed using RT-PCR and western blot analyses. To examine the effects of TRIM24 on Wnt/β-catenin signaling, the NC-transfected and TRIM24 siRNA-transfected cells were incubated with the Wnt/β-catenin signaling activator, lithium chloride (LiCl; 20 mmol/l) for 24 h.

An MTT assay was used to assess cell proliferation, according to the manufacturer's protocol. Briefly, the NC-transfected and TRIM24 siRNA-transfected cells were seeded into 96-well plates at a density of 1×10³ cells/well. Following various durations (1–5 days), 20 µl MTT (Sigma-Aldrich; Merck Millipore) was added to each well and incubated at 37°C for 4 h; dimethyl sulfoxide (150 µl; Sigma-Aldrich; Merck Millopore) was mixed into each well for 10 min, and the absorbance at 490 nm was measured using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Each sample had four replicates.

The NC-transfected and TRIM24 siRNA-transfected MGC803 and HGC-27 cells (3×10² and 4×10²/well, respectively) were seeded in four 6-cm dishes and cultured for 2 weeks. Colonies (>50 cells) were visualized using 5% crystal violet staining and counted. The results are reported as the average of three independent experiments.

The cells were collected 48 h following siRNA transfection, and cell cycle phase was determined using flow cytometry (Beckman-Coulter, Inc., Fullerton, CA, USA). The NC-transfected and TRIM24 siRNA-transfected cells were washed in cold PBS and fixed overnight in 1 ml of 70% ethanol. The following day, the cells were collected, washed and stained with propidium iodide (PI) for 30 min at 4°C. Apoptosis was quantified using an Annexin V-Fluorescein Isothiocyanate Apoptosis Detection kit (Sigma-Aldrich; Merck Millipore) according to the manufacturer's instructions. Briefly, 1×10⁶ NC-transfected and TRIM24 siRNA-transfected cells were collected 48 h following transfection, washed twice in cold PBS, resuspended in 500 µl binding buffer, incubated with Annexin V-PI for 15 min at room temperature and analyzed using flow cytometry.

The cells were seeded into 24-well Transwell plates with a pore size of 8 µm (Corning Incorporated, Corning, NY, USA). The upper chamber was either left uncoated for the migration assay or precoated with Matrigel for the invasion assay. For the migration assay, the MGC803 (6×10⁴) and HGC-27 (5×10⁴) cells were seeded into the upper chamber; for the invasion assay, 1×10⁵ MGC803 and 1×10⁵ HGC-27 cells were seeded into the upper chamber, respectively. The upper chamber was filled with RPMI 1640 medium containing 10 g/l BSA (Sigma-Aldrich; Merck Millipore) and the lower chamber was filled with RPMI 1640 containing 25% FBS. Following incubation for 24 or 36 h at 37°C, the cells that had invaded into the lower chamber were fixed with 4% paraformaldehyde and stained with crystal violet for 1 h at room temperature, and counted in five randomly-selected microscopic fields. All these experiments were performed in triplicate.

The cells were cultured in confocal dishes and fixed in 4% paraformaldehyde for 20 min washed three times with PBS and then permeabilized with 0.2% Triton X-100 for 10 min, all at room temperature. Following blocking with 5% BSA at room temperature for 2 h, the cells were incubated with rabbit anti-human monoclonal anti-β-catenin antibody (1:200 dilution; catalog no. 8480; Cell Signaling Technology, Inc.) overnight at 4°C. Following washing in PBS three times, the cells were incubated with appropriate Alexa Fluor 488-conjugated secondary antibodies (1:500 dilution; catalog no. ab150077; Abcam) for 1 h at room temperature, and then counterstained with 4′,6-diamidino-2-phenylindole for 10 min. Fluorescence images were captured using laser confocal microscopy.

Data were analyzed using paired t-tests to compare quantitative variables. Differences in the expression of TRIM24 between tumor and normal tissues were compared using the Mann-Whitney U test. χ² tests were used to assess the correlation between the expression of TRIM24 and clinicopathological characteristics. Survival curves were plotted using the Kaplan-Meier method, and OS rates were compared using the log-rank test. All analyses were performed using SPSS version 18.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To investigate the role of TRIM24 in the progression of GC, the present study examined the protein levels of TRIM24 in five human GC cell lines (AGS, BGC823, MGC803, HGC-27 and SGC7901) and one normal gastric cell line (GES-1). TRIM24 was expressed in all cell GC lines, being particularly high in the MGC803 and HGC-27 cells, and low in the AGS cells, compared with the level in the GES-1 cells (Fig. 1A). The expression of TRIM24 was then randomly detected in four pairs of GC tissues and adjacent non-tumor tissues, it was found that the expression of TRIM24 was higher in the tumor tissues, compared with the adjacent non-tumor tissues (Fig. 1B). The present study subsequently performed immunohistochemistry on 60 normal gastric tissues and 90 primary GC tumor tissues to detect the expression levels of TRIM24. It was found that TRIM24 was expressed in the nuclei of the tumor cells, whereas the normal tissues exhibited negative or weak nuclear staining (Fig. 1C). A significant upregulation in the expression of TRIM24 was observed in 58.9% (53/90) of the GC tissues, compared with the normal tissues, and this difference was statistically significant (P<0.001; Fig. 1D).

The present study investigated the association between the expression of TRIM24 and the clinicopathological features of GC (Table I). A high expression level of TRIM24 was correlated with depth of invasion (P=0.007), tumor-node-metastasis (TNM) stage (P=0.005) and lymph node metastasis (P=0.027). However, no correlations were found between the expression of TRIM24 and other variables, including age (P=0.848), gender (P=0.294), tumor size (P=0.571), location (P=0.605), tumor differentiation (P=0.176) or Lauren classification (P=0.551). Kaplan-Meier analysis revealed that OS rates were lower in patients with high levels of TRIM24, compared with those with low levels (P=0.010; Fig. 1E; log-rank test). These data indicated that TRIM24 was overexpressed in GC cell lines and tissues, which was associated with a more malignant phenotype and poorer prognosis in patients with GC.

To investigate the function of TRIM24 in GC, the present study evaluated the effects of TRIM24 knockdown on the proliferation and apoptosis of MGC803 and HGC-27 cells. The mRNA and protein levels of TRIM24 were decreased following transfection with TRIM24 siRNA (Fig. 2A and B). TRIM24 knockdown inhibited proliferation, as determined using the MTT and colony formation assays (Fig. 2C and D). Flow cytometric analysis revealed a decrease in the percentage of cells in the S phase and an increase in the percentages of G1/G0 phase cells, suggesting that TRIM24 knockdown induced G1/S arrest (Fig. 2E). Moreover, TRIM24 knockdown increased early apoptosis in the MGC803 (10.4%) and HGC-27 (14.2%) cells, compared with the control MGC803 (4.2%) and HGC-27 (6%) cells (Fig. 2F). These results indicated that TRIM24 had an oncogenic role in the GC cells.

As the expression of TRIM24 was correlated with lymph node metastasis, the present study performed Transwell migration and invasion assays to evaluate the role of TRIM24 in these processes. TRIM24 knockdown inhibited the migration and invasion of MGC803 and HGC-27 cells, compared with the control cells (Fig. 3A and B), demonstrating that TRIM24 promoted GC cell migration and invasion.

Aberrant activation of the Wnt/β-catenin signaling reportedly contributes to the development and progression of GC (2,24). Therefore, the present study examined the association between the expression of TRIM24 and β-catenin in patients with GC. It was found that β-catenin was accumulated predominantly in the nuclei of the GC cells, and a higher level of TRIM24 was associated with elevated nuclear expression of β-catenin (Fig. 4A). To further investigate the correlation between TRIM24 and β-catenin, immunohistochemical analysis was used to detect the expression of TRIM24 and β-catenin in the same sample. Correlation analysis revealed that TRIM24 was positively associated with the nuclear expression of β-catenin; β-catenin positive staining was observed in 43 of the high TRIM24-expressing tumors, but only in nine of the low TRIM24-expressing tumors (P<0.001; r=0.566; Fig. 4B).

To clarify the role of Wnt/β-catenin signaling in the TRIM24-mediated progression of GC, the present study examined the expression levels of downstream genes in the Wnt/β-catenin signaling pathway following TRIM24 knockdown using RT-qPCR and western blot analyses. The knockdown of TRIM24 resulted in the downregulation of β-catenin, cyclinD1 and c-Myc at the mRNA and protein levels (Fig. 4C and D). Furthermore, TRIM24 knockdown significantly reduced nuclear β-catenin accumulation in MGC803 and HGC-27 cells, compared with the control groups, as detected using immunofluorescent staining (Fig. 4E), indicating that the knockdown of TRIM24 inhibited the Wnt/β-catenin pathway through preventing the nuclear translocation of β-catenin.

To further confirm the above findings, LiCl, which inhibits GSK-3β activity, was used to activate Wnt/β-catenin signaling in GC cells. LiCl treatment induced the upregulation of β-catenin, cyclinD1 and c-Myc, and also abrogated the effects of TRIM24 knockdown on the Wnt/β-catenin pathway (Fig. 4F). These data suggested that TRIM24 functioned as a positive regulator of Wnt/β-catenin signaling, which consequently resulted in an aggressive phenotype in GC.