This study was approved by the ethics committee of Xuanwu Hospital, Capital Medical University in Beijing. A written informed consent approved by the ethics committee, was obtained for each patient prior to tissue specimen collection.
Cell Culture, 5-aza-2'-deoxycytidine (DAC)/trichostatin A (TSA) Treatment, and Tissue Samples
Human gastric cancer cell lines (AZ521, AGS and MKN28) were obtained from China Center for Type Culture Collection (Wuhan, China). Cell lines were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin at 37°C in a humid incubator with 5% CO2. To analyze restoration of EMX2 gene, cells were treated with 4 µM DAC or 300 nM TSA (Sigma, St. Louis, USA) for 4 days, with replacement of fresh medium containing the same dose of DAC or TSA everyday. Control cells were cultured with fresh medium containing DMSO. Cells were consequently harvested for DNA/RNA extraction.
A total of 20 formalin-fixed paraffin-embedded tissues blocks (10 gastric cancer tissues and 10 adjacent normal tissues), as well as total RNA from 15 gastric dysplasia samples and 20 gastric cancer surgical samples were obtained from Xuanwu Hospital, Capital Medical University in Beijing. Total RNA and genomic DNA extracted from human adult normal gastric tissues were purchased from Biochain (Hayward, CA, USA).
Topflash/Fopflash reporters containing wild type and mutant TCF/LEF binding sites respectively were kindly provided by Dr. Yeguang Chen (Tsinghua University, Beijing, China). A mutant CTNNB1 (S45Y) cDNA construct was also kindly provided by Dr. Yeguang Chen (Tsinghua University, Beijing, China). Recombinant adenovirus vectors expressing EMX2 and the control vector were purchased from Vector Biolabs (Vector Biolabs, Burlingame, CA, USA).
Methylation-specific PCR (MSP) and Bisulfite Sequencing (BS)
The bisulfite conversion from tissues and cells were performed without the prerequisite for DNA purification using EZ DNA Methylation-Direct Kit (Zymo, Orange, CA, USA). The FFPE tissues were first de-paraffinized in xylene (Sigma) and rehydrated in graded ethanol, before bisulfite treatment per manufacture’s instructions. For MSP, modified DNA is amplified using MSP primers (listed below) which specially recognized either the methylated or unmethylated EMX2 promoter sequences after bisulfite treatment. PCR was run in a final volume of 20 µl containing 1×MSP reaction buffer 
, 0.5 µM of each primer and 1 U of ZymoTaq
™ DNA Polymerase (Zymo). PCR condition was as follows: 10 min at 95°C; 40 cycles of 30 s at 95°C, 30 s at 50°C and 30 at 72°C; and 7 min final extension at 72°C. For BS, amplification of bisulfite-converted DNA was performed in a final volume of 50 µl containing 1 µM each BS primer and 2 U of ZymoTaq
™ DNA Polymerase. PCR condition was as follows: 10 min at 95°C; 40 cycles of 30 s at 95°C, 40 s at 55°C and 1 min at 72°C; and 7 min final extension at 72°C. The PCR products were cloned into pMD-18T (Takara, Dalian, China). Either 5 or 3 randomly selected positive clones from each group were sequenced in Shanghai Invitrogen (Shanghai, China). Primers were as follows:
MSP-M (forward): 5′- TAGTTTTTTGTTCGTTTCGCGTTTC-3′
MSP-M (reverse): 5′- GAATTAAAATAAACGCCCCTACCGAC-3′
MSP-U (forward): 5′-GTTTTTTGTTTGTTTTGTGTTTTGA-3′
MSP-U (reverse): 5′-CCAAATTAAAATAAACACCCCTACCAAC-3′
BS-A (forward): 5′-GTTTGTAAATTTTTTTGGAAGGATTT-3′
BS-A (reverse): 5′-AACAAAAAACTATCCTTAACCACCA-3′
BS-B (forward): 5′-GGTTAGGGATTTTGTAGGGAT-3′
BS-B (reverse): 5′-AAAATCACATAAACAACTTCCTCC-3′
IHC was performed following standard protocol. Antibodies used in the study included mouse anti-human Ki67 (1
100; BD, San Jose, CA, USA) and mouse anti-human EMX2 (1
500; Pierce, Rockford, IL, USA), and Alexa 594-conjugated goat anti-mouse secondary antibody (1
200; Invitrogen, Carlsbad, CA, USA). Slides were also counterstained with hochest 33342 (Invitrogen). Zeiss LSM 710 confocal microscope (Oberkochen, Germany) was used to examine the staining. The intensity of Ki67 was quantified using Image Pro® Plus. EMX2 staining was visualized with Histostain Plus DAB kit (broad spectrum, Invitrogen) according to manufacture’s instructions, counterstained with hematoxylin (Sigma), and photographed using a Leica SCN400 slide scanner (Leica, Germany).
Cells at a density of 5×103 per well were seeded into 24-well plates before transfection. Topflash or Fopflash plasmids were co-transfected with PRL-TK plasmid. After cells were cultured for 18 hours, the luciferase activity was measured by Dual-Glo Luciferase Assay System (Promega, Madison, USA). The ratio between firefly luciferase activity (Topflash/Fopflash) and renilla activity was used for TCF/LEF transcription activity.
Both total protein (20 µg) and cytoplasm extracts (40 µg) were subjected to immunoblotting. The primary antibodies included anti-EMX2 (1
500; Pierce), anti-β-Actin (1
5000; Sigma), anti-Cyclin D1 (1
500; BD) and anti-c-Myc (1
200; Santa Cruz Biotechnology, CA, USA).
Cell growth was determined by CellTiter 96 Aqueous Proliferation Assay Kit (Promega). The cell lines were plated into 96-well culture plates with the number of 5×102 cells/well. After the cells were cultured for days 0, 2, 3 and 4, the MTS solutions were added to the medium and incubated for 1.5 h. The absorbance at 490 nm was measured using microplate reader model 680 (Bio-Rad, Hercules, CA, USA).
Colony Formation Assay
Cells (1×103) infected with adenoviral vector expressing EMX2 or empty vector were seeded in triplicate into 100-mm dishes. Fresh medium was changed every 3 days. After cultured for 3–4 weeks, cells were fixed with 4% paraformaldehyde for 10 min, washed with PBS, stained with 0.1% crystal violet for 20 min and photographed.
RNA Extraction and Quantitative Real-time Reverse Transcription-PCR (RT-PCR)
RNA extraction and real-time RT-PCR were performed as previously described 
. The primer sequences were previously described 
Animal Model and Adenoviral Vector Delivery
All mice experiments were conducted under specific pathogen-free conditions in animal facility of Tsinghua University and approved by the Institutional Animal Care and Use Committee of Tsinghua University. Gastric cancer xenografts were established into 5-week-old female BALB/c nude mice. Briefly, after cultured to confluence, MKN28 or AGS cells were trypisnized and resuspended in PBS (pH 7.4) and then mixed 1
1 (v/v) with matrigel (Vigorous) at 4°C to inject one mouse in a total volume of 150 µl. The mixture was s.c injected into right flank of 16 female mice with 107
cells per mouse (day 0). On day 7, mice bearing local tumors ranged from 50 to 100 mm2
received a direct intra-tumoral injection of 1×109
plaque-forming units of the indicated adenovirus (diluted with PBS in a total volume of 100 µl). The tumor formation was monitored for up to 1½ months. Tumor size was measured using caliper and determined by multiplying by 0.5×width2
×length. The Kaplan-Meier method was used to estimate survival of the two groups of treated animals. At completion of the experiment, the tumors from each treatment were collected in 10% buffered formalin, embedded in paraffin and cut into 5-µm thick slices for further Ki-67 detection. Cytosolic proteins and whole cell lysate were also extracted from those tumors for Western analysis.
All data were calculated as means ± standard deviations. Differences between groups were compared with a two-sided student’s t-test. A P value of 0.05 or less was considered to be significant.