The human GC cell lines AGS and MKN-45 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) was maintained in Dulbecco's modified Eagle's medium (DMEM; HyClone, UT, USA) supplemented with 10% fetal bovine serum (FBS; Tianhang Biological Technology Co., Ltd, Zhejiang, China), penicillin (100 U/ml) and streptomycin (100 µg/ml) (Gibco-BRL, Invitrogen Life Technologies, Inc., Carlsbad, CA, USA) at 37°C in a humidified 5% CO₂ incubator. The pEGFP-N1 plasmid (Clontech Laboratories, Inc., Mountainview, CA, USA) was used to construct the CDX2 expression vector. Total RNA was extracted from human GC AGS cells using TRIzol^® (Invitrogen Life Technologies, Inc.). The resulting RNAs were treated with RNase-free DNase (Promega Corporation, Madison, WI, USA) and 800 ng RNA was reverse transcribed into cDNA using the PrimeScript RT Reagent kit (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's instruc tions. CDX2 cDNA was amplified using a reverse transcription-polymerase chain reaction (RT-PCR) kit (Invitrogen Life Technologies, Inc.), according to the manufacturer's instructions. The complete cDNA sequence of CDX2 was amplified by RT-PCR using primers, which were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The sequences of the primers were as follows: Forward 5′-TTTTCTCGAG ATGTACGTGAGCTACCTCCTG-3′, which introduced an XhoI cleavage site sequence (CTGCTC), and reverse 5′-TTTT AAGCTTCTGGGTGACGGTGGGGTTTAG-3′ which introduced a HindIII cleavage site sequence (CTTAAG). The CDX2 cDNA PCR product and pEGFP-N1 were digested using XhoI/HindIII (Fermentas, Thermo Fisher Scientific, Inc., Vilnius, Lithuania). The PCR amplification (Bio-Rad T100, Bio-Rad Laboratories, Inc., Hercules, CA, USA) conditions were as follows: Initial denaturation at 94°C for 2 min, followed by 25 cycles of 94°C for 60 sec, 55°C for 60 sec and 72°C for 2 min, followed by a final extension step at 72°C for 5 min. Subsequently, 5 µl PCR products were electrophoresed on 1% agarose gel. The PCR products were purified using the DNA Purification kit (Beyotime Institute of Biotechnology, Nantong, China), according to the manufacturer's instructions, and ligated into the cut vector to form pEGFP-N1-CDX2. Following ligation, the plasmid was transformed into E. coli DH5α cells (Takara Biotechnology Co., Ltd.), and plated on solid lysogeny broth medium (containing kanamycin; Yaopharma Co., Ltd., Chongqing, China). Kanamycin-resistant colonies were cultured at 37°C overnight with agitation. The recombinant plasmid was prepared, and the sequences were verified by electrophoresis of the digested product. pEGFP-N1-CDX2 was transfected into GC MKN-45 cells using Lipofectamine™ 2000 reagent (Invitrogen Life Technologies, Inc.) according to the manufacturer's instructions. Briefly, 24 h prior to transfection, cells were seeded into six-well plates (5×10⁵ cells/well) and inoculated in complete medium without antibiotics (2 ml/well). At 70–80% confluence, the plasmid DNA (4.0 µg/well) and transfection reagent (10 µl/well) were diluted with RPMI-1640 (250 µl) and incubated at room temperature for 5 min. The transfection reagent and dilution of the plasmid DNA were then mixed and incubated at room temperature for 20–30 min. Next, the transfection complexes were filled into six-well plates, supplementary medium was added at 2 ml/well and plates were cultured at 37°C in a humidified 5% CO₂ incubator. Four to six hours after transfection, the supernatant was replaced with DMEM containing 10% FBS followed by further cultivation.

The total RNA from cultured cells was extracted using the TRIzol^® reagent extraction kit (Invitrogen Life Technologies, Inc.) according to the manufacturer's instructions. Amplification was performed in a 7500 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) using the QuantiTect SYBR Green PCR kit (Qiagen, Hilden, Germany). PCR was performed as previously described using primers specific for β-actin and CDX2 (20). The β-actin gene was adopted as an internal control for all RT-qPCR reactions.

The cells were lysed using radioimmunoprecipitation buffer and phenylmethylsulfonyl fluoride (all Beyotime Institute of Biotechnology) for 30 min on ice. The protein extracts were subsequently centrifuged at 12,800 x g for 20 min at 4°C, and the protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Equal amounts (40 µg) of protein from various samples were separated by 12% SDS-PAGE (Beyotime Institute of Biotechnology) and electro-transferred onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA) using the Mini Protean 3 system (Bio-Rad Laboratories, Inc.). PVDF membranes were blocked with phosphate-buffered saline (PBS) containing 5% skimmed milk powder for 2 h and incubated with the following primary antibodies: Mouse anti-CDX2 monoclonal antibody (cat. no. 60243-1-Ig), rabbit anti-E-cadherin polyclonal antibody (cat. no. 20874-1-AP) and rabbit anti-vimentin polyclonal antibody (cat. no. 10366-1-AP) (1:2,000; all Proteintech Group, Inc., Chicago, IL, USA), as well as the internal control mouse anti-β-actin monoclonal antibody (1:2,000; cat. no. M20010; Abmart, Inc., Shanghai, China) at 4°C overnight. The membranes were then incubated for 2 h at room temperature with horseradish peroxidase-conjugated anti-mouse (cat. no. 115-032-003) or anti-rabbit (cat. no. 111-035-003) immunoglobulin G secondary antibodies (Jackson ImmunoResearch Europe Ltd., Newmarket, UK), diluted at 1:5,000. The protein was visualized using an enhanced chemiluminescent detection system, according to the manufacturer's instructions (GE Healthcare Life Sciences, Little Chalfont, UK). Following chemiluminescence, the membrane was exposed to X-ray film (Eastman Kodak, Rochester, NY, USA), and ImageJ 1.37 software (National Institutes of Health, Bethesda, MD, USA) was used for grey value analysis. Relative amounts of proteins were quantified by absorbance analysis, and the level was normalized to β-actin.

Cell proliferation was determined using a CCK-8 (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Briefly, cells (5×10⁷ cells/l) suspended in DMEM (100 µl) containing 10% FBS were seeded in 96-well plates and incubated for 24, 48 or 72 h. CCK-8 solution (10 µl) was added to each well and the cultures were incubated at 37°C and 5% CO₂ for 2.5 h. The absorbance was determined at 450 nm using a multi-mode microplate reader (Varioskan Flash; Thermo Fisher Scientific).

The colony formation assay was performed as described previously (21). Briefly, 100 cells were seeded onto each of the 35-mm dishes and allowed to grow for two weeks to assess colony formation on the culture plates. Cell colonies were stained with 0.5% crystal violet (Amresco LLC, Solon, OH, USA) for 5 min at room temperature. Colonies of >50 cells were counted under an upright light microscope (BX51; Olympus Corporation, Tokyo, Japan; magnification, ×100) and the mean value was calculated.

For the cell migration ability assays, 5×10⁵ cells transiently transfected for 24 h in the logarithmic growth phase were seeded onto six-well plates. The cells were cultured to form confluent cell monolayers and then wounded by performing a horizontal scratch with a pipette tip. Non-adherent cells were gently removed by rinsing with PBS. Wound closure was monitored using an inverted fluorescence microscope (IX71; Olympus Corporation; magnification, ×100) at various time-points. Migration activity was calculated as the mean distance between the edges of three points. Healing rate = (mean original distance - mean distance at a time-point)/mean original distance ×100%.

For the invasion assays, 1×10⁵ cells transiently transfected for 48 h were seeded in the top chamber of a Transwell plate containing a Matrigel-coated membrane (24-well insert; 8 mm pore size; BD Biosciences, Franklin Lakes, NJ, USA). Serum-free medium was used for the cells in the upper chamber containing the cells, and medium supplemented with 20% (v/v) FBS was used as a chemoattractant in the lower chamber. The cells were incubated for 24 h at 37°C and in a 5% (v/v) CO₂ incubator. Following incubation, the non-invading cells were removed from the upper side of the Transwell membrane filter inserts using cotton-tipped swabs. The invaded cells on the lower side of the inserts were stained with Giemsa, according to the manufacturer's instructions (Wright-Giemsa Stain kit; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Visual fields (n=15) of each insert were randomly counted under an upright light microscope (BX51; Olympus Corporation) and the mean value was calculated.

The present study was approved by the ethics committee of the Affiliated Hospital of Nantong University (Nantong, China). The experiments were performed according to the Nantong University Institutional Animal Care and Use Committee. To analyze the tumorigenicity of GC cells in vivo, MKN45 cells at twenty-four hours after transfection were diluted to 1:4 for passage and MKN45/CDX2 stable cell lines were screened by administration of G418 (Invitrogen Life Technologies, Inc.). Four-week-old male or female BALB/c nu/nu nude mice were purchased from the Center of Laboratory Animal Science, Chinese Academy of Medical Science (Shanghai, China). According to the experimental animal guidelines, the mice were maintained in 24°C temperature-controlled and specific pathogen-free conditions; they were housed separately, and were fed sterilized food and autoclaved water. The mice were subcutaneously injected in the dorsal flanks with 2×10⁷ MKN45/CDX2, empty-vector (EV) transfected MKN45/EV or native MKN45 cells, respectively, suspended in 200 µl PBS. Mice were weighed, and tumor width and length were measured with calipers three times a week. Mice were sacrificed at seven weeks after cell inoculation. Tumor volume was calculated using the formula V (mm³) = (width² × length)/2. The mice were sacrificed by cervical dislocation 7 weeks after cell inoculation, and the tumor tissue samples were taken to assess the protein expression levels of vimentin and E-cadherin. The tumor tissues were homogenized using an ultrasonic processor (S-450D; Branson Ultrasonics Corporation, Danbury, CT, USA) for tissue lysate extraction, the tissue lysates were centrifuged at 12,800 × g for 20 min at 4°C and the supernatants were collected. Equal quantities (150 µg) of protein were loaded per lane for western blot analysis.

All experiments were repeated at least three times. Statistical analysis was performed using SPSS 19.0 (SPSS, Inc., International Business Machines, Armonk, NY, USA). Values are expressed as the mean ± standard deviation and groups were compared using one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

A previous study showed that the expression of CDX2 on MKN-45 cells was low (19). To examine the effects of CDX2 on gastric cancer cells in vitro, the plasmid pEGFP-N1 was used to construct the CDX2 expression vector, pEGFP-N1-CDX2, to be transfected into the MKN45 cells to create the engineered cell line MKN45/CDX2 with forced overexpression of CDX2 protein. A control cell line was transfected with the empty vector and named as MKN45/EV. The transfection efficiency was confirmed by fluorescence microscopy observation. The results showed that >80% cells were labeled with EGFP, indicating that MKN45 cells had been successfully transfected with pEGFP-N1-CDX2 (Fig. 1A and B). mRNA and protein levels of CDX2 in MKN45 cells were validated by RT-qPCR and western blot analysis, respectively. Compared with the control groups (MKN45/EV and MKN45), CDX2 mRNA and protein expression were significantly enhanced in MKN45/CDX2 cells (P<0.05; Fig. 1C and D).

To investigate the effects of CDX2 overexpression on the growth of MKN45 cells, cell viability was assessed using the CCK-8 assay. The results showed that high expression of CDX2 in the MKN45/CDX2 group significantly inhibited the cell proliferation compared with that in the MKN45/EV group and the untransfected MKN45 group (P<0.05; Table I). These results were further supported by the colony formation assay. The three cell lines were all able to form colonies in soft agarose; however, the number of colonies formed by MKN45/CDX2 cells after two weeks of culture was significantly decreased compared with that in the MKN45/EV and MKN45 cells (P<0.05; Fig. 2A and B).

The scratch-wound assay was used to compare the scratch width at 0, 24, 48 and 72 h. The results showed that MKN45/CDX2 cells had a decreased migratory ability at the 48 and 72 h time-points compared to that of the control groups MKN45/EV and MKN45 (P<0.05; Fig. 3A). Furthermore, the invasion ability of three groups was assessed using a Transwell^® assay. The number of cells that had transgressed through the membrane was counted following transfection for 48 h. The results showed that the numbers of MKN45/EV and MKN45 cells that had invaded through the membrane of the Matrigel chamber were ~2.3-fold higher than that in the MKN45/CDX2 group (P<0.05; Fig. 3B).

To study the effects of CDX2 on the EMT of MKN45 cells, the expression of E-cadherin and vimentin was determined using western blot analysis. MKN45/CDX2 cells overexpressing CDX2 exhibited a significant upregulation of E-cadherin protein expression and a significant downregulation of vimentin protein expression (P<0.05; Fig. 3C).

To assess the pathophysiological role of CDX2 in gastric tumor xenograft growth, in vivo tumorigenicity assays were performed, which showed that mice injected with MKN45/CDX2 cells developed smaller and slower-growing tumors when compared to those in animals injected with the control MKN45/EV and MKN45 cells (P<0.05; Fig. 4A and B; Table II). Furthermore, western blot analysis of the tumor tissue showed that MKN45/CDX2 cells exhibited a significant upregulation of E-cadherin protein and a significant downregulation of vimentin protein in nude mice (P<0.05; Fig. 4C).