| Background and objective: Gastric cardia adenocarcinoma (GCA) is one of the most common malignant diseases in Northern China. The incidence rate of tumors of distal gastric has descended obviously from 1980s, but the incidence rate of GCA still keeps in high level. It is suggested that there are multiple phases and factors which participate the development of GCA. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) is a new technology of proteomics developed these years and has the characters of tiny doses, convenient operation, high-flux, high sensitivity and high veracity, which can provide a satisfactory technical platform for the studying of GCA. But there are few reports about the studying of proteomics with GCA. IMAC-Cu proteinchip array and SELDI-TOF-MS were used to analyze serum samples of GCA patients and healthy controls. The proteomic pattern was founded to diagnose GCA accurately. The serum of different differentiated degrees of GCA and GCA with or without lymph nodes metastasis were be analyzed to screen related markers and find the rules of changes, which can provide clues for the research about mechanism of cancerization with GCA. On the base of above-mentioned, the candidate proteins related with GCA were screened by using bioinformations. The technology of reverse transcription multiplex polymerase chain reaction (RT-PCR) was used to detect the expression levels of genes (mRNA) of candidate proteins in the tissues of GCA patients to search markers of GCA early diagnosis.Materials and methods: GCA patients, 36 were males and 11 were females, with a mean age of 61±8 years in male and 61±10 years in female. All the patients were diagnosed with gastric cardia adenocarcinoma. Of the all patients, 23 cases were lymph nodes metastasis. Healthy controls: 29 were males and 21 were females, with a mean age of 56±11 years in male and 60±10 years in female. Two-five milliliter blood were obtained from all the cases on an empty stomach, standing in 4℃1 to 2 hours, and centrifuged 30 minutes with 2000r/min, conserved in refrigerator of -80℃. Twenty cases tissue samples (10 cases were GCA tissue and 10 were the normal tissue aside) from the matched patients in our study, of the patients 6 cases were male and 4 cases were female, with a mean age of 60±7 years.A total of 10μ1 of each sample was diluted to 20μl with U9 buffer (9M Urea, 2% CHAPS, 50mM Tris-HCl, PH 9.0) and vortex at 4℃for 30 min. 10μl of the supernatants was mixed with 120μ1 binding buffer (50mM NaAC at PH3.5), and vortex for 5min. An eight-spot IMAC-Cu array was loaded with 50μl 100mM CuSO4, 50μl of 100mM Na Acetate 150μl 50mM HEPES (pH7.0), and 50μl of diluted serum sample was spotted on to each IMAC-Cu array spot and incubated for 60 min in 4℃on a shaker. Then washed with PBS solution (pH7.0). After washing, 0.5μl SPA was applied on each spot and allowed to air-dry. Mass spectrometry was performed in a PBS-Ⅱmass reader (Ciphergen Biosystems). Data were collected between 1500 and 20 000Da. Peaks with M/Z＜1500 were excluded as the energy absorbing matrix signal generally interfered with peak detection in this region. Mass accuracy was calibrated externally using the All-in-1 peptide and All-in-1 Protein molecular mass standards (Ciphergen Biosystems). CipherGen ProteinChip software was used to read the data. Then the primary data was analyzed to draw the proteomic mass-spectrogram.The retrieval of related proteins: TagIdent tool from the ExPASy molecular biology server (http://us.expasy.org/tools/tagident.html) was used to retrieve the candidate protein based on protein molecular weight. By inputting the mass of an unknown protein, this tool will search in the SWISS-PROT and TrEMBL protein databases for proteins that could match with the requested mass. The extent of mass error was less than 0.5 %.RT-PCR: Abstraction of RNA was followed by introductions of SV Total RNA Isolation System. Reverse transcription was followed by introductions of ImProm-IITM Reverse Transcription System. The primer was composed by the biological company in Shanghai. Gastric cancer-related protein (VRG118) forward: 5'-GGC TCC ATG GTC TGA GTT GT-3', reverse: 5'-TTA ATA CCA CTC CCC CAA CG-3',the length was 194bp. The reaction system was 10×Reaction buffer 2μl, 10mmol/L dNTPs 2μl, 25mmol/L MgCl2 1.6μl, forward primer 0.8μl, reverse primer 0.8μl, Taq DNA Polymerase 0.1μl, Template DNA 2μl, Sterile deionized Water 10.7μl. Reaction condition: the PCR procedure consisted of 30 cycles of denaturation at 94℃for 30s, annealing at 60℃for 35s, and extension at 72℃for 45s, with initial denaturation of sample cDNA at 95℃for 3 min and an additional extension period of 8 min after the last cycle.Statistics: All the data were corrected by the Proteinchip Software 3.1 to make the ionic strength and molecular weight to be uniform. Then the Biomarker Wizard (BMW) software application (Ciphergen Biosystems) was used to autodetect M/Z peaks with a signal-to-noise ratio of at least 5. For validation purposes, peak clusters of the training set were applied in the validation set. Group differences were calculated with the same application, comparing mean intensities of all detected peaks between groups with non-parametric statistical tests. P values less than 0.05 were considered statistically significant. Chi-squared test and Kappa test were performed to compare the difference between different groups.Results: Markers pattern made up by proteins (M5908.48, M7943.64 and M8938.70) was able to discriminate the GCA group and control group. The veracity, sensitivity and specialty were 77.3 %, 85.1% and 70% respectively. The content of up-regulated proteins (M4211.29, M5334.13, M5966.63, M5903.14, M11735.71, M5922.70, M7932.54, M7758.66, M9287.40 and M6109.99) in control group, moderately differentiated group and poorly differentiated group had an ascending tendency. The content of down-regulated proteins (M8992.89, M8158.48, M4495.96, M8924.27, M9434.40, M5099.02, M6661.72, M6222.12 and M6960.60) in normal group, poorly differentiated group and moderately differentiated group had a descending tendency. We found that the content of protein with molecular weight of 7762.68Da in the serum of GCA patients was higher than which in healthy controls. According to the searching result in SWISS-PROT and TrEMBL protein databases we found that the Gastric cancer-related protein (VRG118), with a mass of 7780.87Da,was among the first matches for our request. The results of RT-PCR were as follows: the expression of VRG118 in GCA tissues: the positive rate was 40% (4/10). The mRNA expression for VRG118 was not observed in the normal tissue.Conclusion: The markers pattern made up by tumor markers M5908.48, M7943.64 and M8938.70 can be used to diagnose GCA, which has a high sensitivity and specialty. The difference between GCA patients with poor and moderate differentiation is large which may be related with the malignant degrees of proliferation and invasion. The gene (mRNA) expression of candidate protein (VRG118) in the GCA tissues detected by RT-PCR is conformed with the results in serum detected by SELDI-TOF-MS. The results suggest that VRG118 is closely related with the carcinogenesis and development of GCA and can be made as tumor markers of GCA.
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