A total of 108 individuals’ serum samples (50 ESCC patients and 58 risk-matched controls) were collected from Baoding Tumor Hospital, Hebei, China. About 10 g of ESCC tumor samples were also collected in parallel. In addition, 38 serum samples were collected from patients with gastric cancer from the same hospital. Detailed information on the serum samples is listed in Table . All the clinical samples were collected after informed consents were obtained.
Characteristics of normal and patient serum n (%)
Cloning the MMP-7 cDNA from ESCC tissues
About 10 g of each ESCC tissue sample was snap-frozen in liquid nitrogen. RNA extraction was performed according to procedures described in the TRIzol Reagent manual (Invitrogen, USA). Primers were designed according to the sequence of the MMP-7 mRNA (GenBank, NM_002423.3). The sequence of the forward primer was5’-GGAATTCCATATGTCACTATTTCCAAATAGCCC-3’ and the sequence of reverse primer was 5’-CCCAAGCTTTTATCCATATAGTTTCTGAATGCC-3’. NdeI and Hind III (underlined) restriction sites were introduced into the sequences of forward and reverse primers, respectively.
RT-PCR reactions were carried out under the following conditions. After heating at 94°C for 4 min, the reactions were exposed to 30 cycles of 94°C for 30 s, 63°C for 40 s, and 72°C for 1 min; with a final extension at 72°C for 10 min. The final products were then subjected to electrophoresis on 1% agarose. The amplified inserts were purified using a DNA purification kit (QIAGEN, USA), digested with NdeI and Hind III, and then ligated to a prokaryotic expression vector pET28b(+) (Novagen, USA) that was also digested with the same restriction enzymes. The constructed plasmid was transformed into competent E. coli. DH5α cells and grown in Luria-Bertani (LB) broth supplemented with kanamycin (30 μg/mL). The recombinant plasmid was confirmed by double endonuclease digestion and DNA sequencing.
MMP-7 protein expression and purification
The recombinant plasmid pET-28b/MMP-7 was transformed into expression strain E. coli Rosetta (DE3) cells by heat-shock. One colony was picked and grown in 20 mL LB medium containing 30 μg/mL kanamycin at 37°C until an optical density (OD) at 600 nm of 0.6 was reached. Isopropyl-β-D-Thiogalactopyranoside (IPTG) was then added to induce protein expression at 28°C and 37°C. To determine the optimal condition for MMP-7 expression, DE3 cells were induced at different concentrations (0.4, 0.8, and 1.0 mmol/L) of IPTG for different lengths of time (1, 2, 4, 6 and 7 h). At the end of each condition, cells were harvested by centrifugation, resuspended in 1 mL PBS, and sonicated on ice until the suspension became transparent (5 min). The lysate was centrifuged for 30 min at 12 000 g, and then both the supernatant and the pellets were tested for the MMP-7 protein expression by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
To purify this His-tagged MMP-7 recombinant protein, a QIAexpressionist (QIAGEN, USA) kit was used. Briefly, 3 mL of cell culture at the most optimal expression conditions was pelleted and resuspended in 600 μL lysis buffer (8 mol/L Urea, 10 mmol/L NaH2PO4, 10 mmol/L Tris.Cl, pH8.0) at room temperature for 4 h. Cell debris was cleared by centrifugation, and the supernatants were transferred to a fresh tube and incubated with 60 μL of a 50% slurry of Ni-NTA resin (10 μL resin has a capacity for 50-100 μg His-tagged protein) for 60 min at 4°C with agitation. The resin was then pelleted by centrifugation and washed twice with 300 μL wash buffer (8 mol/L Urea, 100 mmol/L NaH2PO4, 10 mmol/L Tris.Cl, pH6.3). The protein was then eluted three times with 30 μL elution buffer (8 mol/L Urea, 100 mmol/L NaH2PO4, 10 mmol/L Tris.Cl, pH4.5). The purification process was tested by 15% SDS-PAGE followed by Coomassie Brilliant Blue staining.
Measurement of serum autoantibodies against MMP-7
Ninety-six-well Costar ELISA plates (Jet Biofil, Beijing, China) were coated with 2 μg/mL of the purified MMP-7 protein and incubated overnight at 4°C. The plates were washed four times with PBST (PBS buffer containing 0.05% Tween 20), and then blocked with PBS containing 1% BSA at 37°C for 1 h, followed by four washes in PBST. Serum samples (ESCC or gastric cancer patients or control) were diluted 1/150 in 1% BSA and incubated in the MMP-7-coated ELISA plates at 37°C for 1 h. After washing four times with PBST, 100 μL of goat anti-human IgG-HRP (1:1000 dilutions) was added to each well for 1h at 37°C. After washing four times with PBST, the color was developed with 3,4,5-trimethoxy benzaldehyde (TMB) for exactly 15 min, and then stopped with 0.5 mol/L H2SO4. The absorbance of each well was read at 450 nm by a plate microplate reader (Beijing’s New Air Electrical Technology, Beijing, China). Each serum sample was tested in triplicate.
To analyze the difference of autoantibodies reaction to MMP-7 proteins between cancer and matched control sera, the absorbance of each serum sample in the ELISA plate was averaged from triplicate experiments. Student’s t-test was performed between cancer and matched-control samples. Nonparametric receiver-operating curves (ROCs), in which the value for sensitivity was plotted against false-positive rate (1-specificity), were generated. In addition, an area under the ROC curve (AUC) with 95% confidence intervals (CI) was calculated for each marker. In all tests, a P-value of ≤ 0.05 was considered to be statistically significant. All statistical analysis was done with the SPSS software package version 16.0 (SPSS, Chicago, IL, USA).