Specific rabbit anti-human polyclonal antibodies to CDX2 (#12306), c-Myc (#5605), Skp2 (#2652), Bax (#5023), Bcl-2 (#2827), cyclin D1 (#2978), survivin (#2808) and GAPDH (#2118) were provided by Cell Signaling Technology, Inc. (Beverly, MA, USA). Infrared-labeled secondary goat anti-rabbit antibodies to IRDye 800 were obtained from Li-Cor Biosciences (Lincoln, NE, USA). All the above antibodies were used by diluted 1000 times in western blot analysis.

The MGC-803 human gastric carcinoma cell line and 293T human embryonic kidney cells were provided by the Cell Bank of Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were maintained at 37°C in an atmosphere containing 5% CO₂ in Dulbecco’s modified Eagle’s Medium, supplemented with 10% fetal bovine serum, 100U/ml penicillin and 100 μg/ml streptomycin.

The lentivitus overexpressing the CDX2 gene was constructed at Shanghai Genechem Co., Ltd. (Shanghai, China). The lentiviral vector system consisted of a GV208, pHelper 1.0 vector and pHelper 2.0 vector prior to packaging and was provided by Shanghai Genechem Co., Ltd. (Shanghai, China). The full length of the human CDX2 gene (NCBI ID, NM_001265.4), which was indicated by enhanced green fluorescent protein (GFP), which was encoded into the GV208 vector. The three vectors were cotransfected into 293T cells (3×10⁴/ml) in serum-free medium using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA). The medium was replaced with complete medium following 8 h incubation at 37°C. The high-titer recombinant lentiviral vectors carrying CDX2 were harvested 48 h following transfection.

The animals used in the present study were BALB/c nude male mice (4 weeks old), which were purchased from Guangxi Animal Center (Nanning, China). The number of nude mice was six in each group, weighing between 20 and 24 g, and they were fed under specific pathogen-free conditions. All procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA). Tumors were established in the mice via a single subcutaneous injection of 4×10⁷ MGC-803 cells into the armpit region. The study was approved by the ethics committee of the First Affiliated Hospital of Guangxi Medical University, (Guangxi, China).

When the tumors had reached a diameter of ~5 mm, the mice were randomized into three groups: Lentivirus (LV)-GFP-CDX2, LV-GFP-negative control (NC) and phosphate-buffered saline (PBS; Beyotime Institute of Biotechnology, Shanghai, China). Each group contained eight mice (n=8). The animals were administered with an intratumoral injection of either the LV-GFP-CDX2 or LV-GFP-NC at a titer of 10⁸ transducing units in 100 μl PBS, while the control group of mice received an equal volume of PBS. Subsequent to the first injection, the animals were administered with a similar injection every 2 days. The mouse body weight, the quantity of water and food intake, vital signs and living status were assessed daily. The tumor volume was measured and calculated as follows: The longer diameter, ‘a’, and the shortest diameter, ‘b’, of the tumors were measured using digital calipers, and the tumor volume (TV) was calculated using the following equation: TV = a × b²/2. The relative tumor volume (RTV) was calculated using the formula: RTV = V_t / V₀, in which V₀ is the TV on the day when the treatment was administered, and V_t is the TV of the subsequent measurement. Following the tumor cell injections (15 days), the animals were sacrificed by cervical dislocation and the tumors were then analyzed.

The total RNA was extracted from the tumor tissues using TRIzol reagent (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer’s instructions. cDNA was generated from a DNase-1-treated RNA template with 0.2 μg random hexamer primers (Takara Bio, Inc., Tokyo, Japan) and 200 units RevertAid H-Minus M-MuLV reverse transcriptase enzyme (Roche, Basel, Switzerland). The primer sequences used to specifically amplify the genes of interest are shown in Table I. The cDNA (2 μl) produced was added to 10 μl Taq Premix and the upstream and downstream primers (1 μl each). RT-qPCR was performed as follows: 1 cycle at 94°C for 5 min, 30 cycles at 94°C for 30 sec for denaturation, 56°C for 30 sec for annealing, 68°C for 45 sec for extension and 1 cycle 5 min at 72°C, according to the RT-qPCR amplification kit (Takara Bio, Inc.) manufacturer’s instructions. The amplified PCR products were run on 1.5% agarose gels and visualized under UV light following ethidium bromide (0.5 μg/ml; Beyotime Institute of Biotechnology) staining at room temperature (25°C) for 20 min.

The tumor tissues were homogenized for tissue lysate extraction, the tissue lysates were centrifuged and the supernatants were collected. Equal quantities (150 μg) of protein were heated to 100°C for 5 min with Laemmli sample buffer (Beyotime Institute of Biotechnology), then separated on 12% SDS-PAGE gels (Beyotime Institute of Biotechnology) and transferred onto polyvinylidene difluoride membranes. The entire process was performed using Bio-Rad equipment (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer’s instructions. The membrane was probed with the primary antibody (1:1,000) and incubated overnight at 4°C. The blots were washed three times in PBS with Tween 20 prior to incubation with species-appropriate, peroxidase-conjugated secondary antibodies for 1 h. The blots were then washed again three times in PBS with Tween 20. The net intensities of the bands were quantified using Odyssey software version 3.0 (Li-Cor Biosciences, Lincoln, NE, USA).

Tissue samples were fixed in 4% buffered paraformaldehyde at 4℃ for 48 h and then processed for paraffin embedding. The procedures of paraffin embedding were dehydration and waxdip. Paraffin-embedded (Beyotime Institute of Biotechnology) sections were prepared for hematoxylin and eosin (Beyotime Institute of Biotechnology) staining. The levels of tumor tissue necrosis were determined by comparing the surface of necrotic areas with that of the whole tumor. Levels of apoptosis were determined using a TUNEL assay kit, according to the manufacturer’s instructions. Briefly, the cells were rinsed with PBS twice for 3 min, prior to the addition of 50 μl TUNEL cocktail on test sections. Labeling solution (40 μl) was added to control sections on one slide and PBS was added to the control sections on other slides, and incubated in a humidified chamber for 60 min at 37ºC in the dark. A sample was considered positive when it contained 25 positively stained cells in every 100 tumor cells, which was calculated from five randomly selected fields for each specimen. The stained tissue sections were visualized by microscopy (CP-111-2; magnification, x400; Jenco International, Protland, OR, USA).

Data are expressed as the mean ± standard error of the mean and were analyzed using SPSS version 13.0 (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance was used to measure statistical significance among groups, followed by the Student-Newman-Keuls test. P<0.05 was considered to indicate a statistically significant difference.

The positive clones were confirmed by DNA sequence analysis (data not shown) and it was demonstrated that the RNA coding frames and frame sequences were correct and that the recombinant pGCL-GFP-CDX2 and pGCL-GFP-NC plasmids had been constructed successfully.

A lentivirus targeting CDX2 and an NC vector (LV-GFP-CDX2, and LV-GFP-NC, respectively) were produced by co-transfection with a packaging vector (pHelper1.0) and a vesicular stomatitis virus glycoprotein expression plasmid (pHelper2.0) into the 293T cells. As shown in Fig. 1, the GFP-labeling results indicated that the lentiviral vectors were suitably transfected for use in the present study.

As shown in Fig. 2A, the tumor growth curves indicated that mice treated with LV-GFP-CDX2 exhibited significant inhibition of tumor growth when compared with those treated with the LV-GFP-NC control vector or PBS (P<0.05). The tumor volumes in the mice in the LV-GFP-CDX2 group were significantly smaller compared with those of the control groups (P<0.05) at 15 days post-tumor injection, whereas no difference was identified between the Lv-GFP-NC and PBS groups (P>0.05; Fig. 2B). These results indicated that overexpression of CDX2 effectively inhibited MGC-803 tumor growth in vivo.

As shown in Fig. 2C, the percentage of apoptotic tumor cells in the LV-CDX2-GFP group was 17.32±2.5%, which was significantly higher compared with that observed in the LV-GFP-NC (7.2±1.7%) and PBS (6.6±1.8%) groups, demonstrated using the TUNEL method (P<0.05). These results suggested that overexpression of CDX2 effectively promoted MGC-803 tumor cell apoptosis in vivo.

Densitometric analysis revealed that mRNA and protein expression levels of CDX2 in the LV-GFP-CDX2 group were higher compared with those of the two control groups (P<0.05; Fig. 3A–D). These results suggested that the nude mouse model overexpressing CDX2 had been constructed successfully by injection with the CDX2 recombinant lentiviral vectors.

As shown in Fig. 4A and B, the densitometric analysis revealed that the mRNA expression levels of c-Myc, Skp2, Bcl-2, cyclin D1 and survivin in the LV-GFP-CDX2 group were lower, while the expression of Bax was higher compared with the LV-GFP-NC and PBS groups (P<0.05). In addition, as shown in Fig. 5A and B, the densitometry revealed that the protein expression levels of c-Myc, Skp2, Bcl-2, cyclin D1 and survivin in the LV-GFP-CDX2 group was lower, that of while Bax was higher compared with the LV-GFP-and PBS groups (P<0.05). These results suggested that the overexpression of CDX2 effectively decreased the expression levels of c-Myc, Skp2, Bcl-2, cyclin D1, survivin, and increased the expression of Bax in the MGC-803 tumor cells in vivo.