A total of fifty-seven primary ESCC patients that underwent esophagectomy were enrolled in this study. Tumor specimens and paired normal esophageal tissue specimens taken from a site distant from the cancerous lesion were obtained from the consenting patients, as approved by the Medical Ethics Committee of Yixing People's Hospital. None of the patients received radiotherapy or chemotherapy before surgery. Clinical and pathological data including age, gender, pathological grading, tumor location, tumor stage and lymph node metastasis were acquired from the medical records.
Human ESCC cell lines Eca109 and TE-1 were purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). Cells were maintained in RPMI1640 (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 100 U/ml penicillin and 100 μg/ml streptomycin, within a humidified atmosphere containing 5% CO2 at 37°C.
We used primary ESCC tissues near the margin of the tumor and match normal tissues to asses PRAF3 expression. Sections (5 μm) of the specimens were incubated with goat anti-human PRAF3 antibody (Santa Cruz) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated donkey anti-goat antibody (Santa Cruz) for 1 hr at 37°C. Immunodetection was performed with the EnVision™ Kit (Dako), using diaminobenzidine as the chromogen. All slides were evaluated independently by two pathologists (ZJG. and JZ.) without prior knowledge of the clinical information of the patients. The expression of PRAF3 was considered positive if staining intensity was moderate or strong and the percentage of positively stained cancer cells were > 10%.
Construction of recombinant adenovirus Ad.PRAF3 and cell infection
Recombinant adenovirus expressing human PRAF3 was constructed using the AdEasy system (Stategene). Human PRAF3 gene was amplified and ligated into pShuttleCMV plasmid. Ad.PRAF3 and Ad.Null were propagated in HEK293 cells and purified using BD Adeno-X™ virus purification kit (BD Biosciences). The activities of adenovirus were determined by plaque assay using BD Adeno-X™ rapid titer kit (BD Biosciences) according to the manufacturer's instruction. Cells from ESCC cell lines were infected with Ad.PRAF3 (or Ad.Null) at a MOI of 100 for 12 hr at 37°C, where the percentage of infected cells were 84.7% and 79.3% as detected by Ad.GFP in Eca109 and TE-1, respectively.
Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA was extracted from 100 mg tissues or 1 × 105 cells using the RNeasy RNA Mini Kit (Qiagen). First strand cDNA was synthesized using POWERSCRIPT reverse transcriptase (Clontech). The following gene-specific primer pairs were used for quantitative PCR:
PRAF3: Forward, 5'- TCATGTTGGCGAGCTATTTCC -3';
Reverse, 5'- GGTTCCGAAGTCTCAACGATG-3'.
GAPDH: Forward, 5'- GCTGAGTATGTCGTGGAGTC -3';
Reverse, 5'- AGTTGGTGGTGCAGGATGC -3'.
PCR was performed using a Fast Start Master SYBR Green Kit (Roche) on a LightCycler (Roche). The expression level of target gene mRNA was analyzed using RealQuant software (Roche) and normalized to that of GAPDH mRNA.
Cell lysis and western blot
For preparation of membrane and cytoplasmic protein samples, the tissue or cell samples were harvested in hypotonic lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 0.2 mM EDTA, 1 mM DTT) supplemented with inhibitors (25 mM b glycerol-phosphate, 25 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 1 mM benzamidine,). Cell lysates were prepared by Dounce homogenisation and centrifuged at 500 g for 5 min to eliminate nuclei and debris. The supernatant was subjected to ultracentrifugation at 20,000 g for 60 min using the TLA-100.2 fixed angle rotor in Optima TL-100 ultracentrifuge (Beckman). The supernatant (cytoplasm) was adjusted to 100 mM NaCl and 0.5% Nonidet P-40. The membrane pellet was resolubilised in NETN buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 200 mM EDTA, 0.5% Nonidet P-40, 1 mM DTT, supplemented with inhibitors).
Equal amounts of protein (50 μg) were separated by 10% SDS PAGE and then transferred to nitrocellulose membranes (NY) by electroblotting. The membranes were blocked with 5% BSA in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.05% Tween 20) for 1 hr, and then incubated with mouse anti-human specific antibodies overnight at 4°C before subsequent incubation with horseradish peroxidase-conjugated goat anti-mouse antibody (BD Biosciences) for 1 hr at 37°C. Protein was visualized using enhanced chemiluminescence reagent (Santa Cruz). The expression level of target protein was analyzed using LabWork 4.0 program (UVP) and normalized to that of β-actin protein.
The activity of MMP-2 and MMP-9 was detected by zymography analysis. The supernatants of ESCC cells (1.0 × 106) were mixed with sample buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 0.2 mM EDTA) without reducing agent or heating. The sample was loaded into a gelatin (1 mg/ml) containing SDS-polyacrylamide gel and separated by PAGE. Then, the gel was washed with 2.5% TritonX-100 to remove SDS, rinsed with 50 mM Tris-HCl, pH 7.5, and subsequently incubated overnight at room temperature with the developing buffer (50 mM Tris-HCl, pH 7.5, 5 mM CaCl2,1 mM ZnCl2, 0.02% thimerosal, 1% Triton X-100). The zymographic activities were revealed by LabWork 4.0 program after staining with 1% Coomassie Blue.
Cell apoptosis was analyzed by annexin V-FITC assay. Briefly, cells were treated with RNase A (50 mg/ml) for 30 min at 37°C and then stained with annexin V-FITC and propidium iodide using the ANNEXIN V-FITC Kit (Beckman) according to the manufacturer's protocol and subjected to flow cytometric analysis. Viable cells were unstained by annexin V or propidium iodide, early apoptotic cells were stained by annexin V but not propidium iodide, and late apoptotic cells were stained by annexin V and propidium iodide.
Caspases activity assay
ApoAlert caspase fluorescent and colorimetric assays (BD Biosciences) were used to measure the activity of caspase-8 and caspase-9. Cells (1.0 × 106) were collected and lysed in ice-cold buffer by homogenating. Caspase activity in the supernatant was determined by cleavage of the specific chromophore-conjugated substrates. The substrate peptides of caspase-8 (IETD) and caspase-9 (LEHD) were conjugated to p-nitroaniline (p-NA), and 7-amino-4-methyl coumarin (AMC), respectively. Caspase-8 activity was determined by absorbance of p-NA at 405 nm in a microplate reader. The release of AMC for caspase-9 was measured by quantifying fluorescent intensity in a fluorescence spectrophotometer.
Wound healing assay
ESCC cells were seeded on 6-well plates at a density of 5 × 105 cells/well. After the cells reached sub-confluence, the mono-layer cells were wounded by scraping off the cells and then grown in medium for 48 h. The migrated distance of cells was monitored and imaged under a microscope. The distances of cell migration were calculated by subtracting the distance between the lesion edges at 48 hr from the distance measured at 0 h. The relative migrating distance of cells is measured by the distance of cell migration/the distance measured at 0 h.
Cell migration and invasion were determined using a transwell (Costar) with a pore size of 0.8 μm. 1 × 105 cells were seeded in serum-free medium in the upper chamber (normal chamber for migration assay and matrigel-coated chamber for invasion assay), while medium containing 10% FBS in the lower chamber. After incubating for 8 hr at 37°C, cells in the upper chamber were carefully removed with a cotton swab and the cells that had traversed to reverse face of the membrane were fixed in methanol, stained with Giemsa, and counted.
Statistical significance was tested using SPSS15.0 software. For comparisons of clinical features except for pathological grading between PRAF3 positive and negative groups, chi-square test was performed. The correlation between PRAF3 expression and pathological grade was analyzed by Cochran-Mantel-Haenszel Statistics. Other data are presented as mean ± SEM, using student t tests for 2-group comparisons. A P value less than 0.05 is considered statistically significant.