Serum Biomarkers Of Gastric Cancer Screening,Identification And The Biological Characteristics Research

1. Background Gastric cancer is a kind of malignant tumor with the highest occurrance rate in the world, which also stays at a high point in China. Compared with foreign cases,more gastric cancer patients in China are diagnosed as being in local metastasis or at advanced stage than early stage, and the survival rate is obviously lower than foreign patients. Thus, how to diagnose and treat gastric cancer has become a hot issue in domestic research now. For lack of obvious clinic symptoms and signs which can also easily be confused with those of gastritis, gastric cancers can hardly be diagnosed until they have been in progressive or advanced stages. However there are still no distinctive clinical features even though the cancer has been in progressive or advanced stage, which makes it apparently difficult to diagnose gastric cancer depending on clinical features. Now, there is still no an effective and simple method to detect gastric cancer earlier in our county. Electronic gastroscope biopsy pathology has been a physical examination item among middle-aged people and the older.However, it is still not popular in China for the high costs, the accompanying pains and latent risks. Some routine measurements such as CEA, CA724, CA-199 can just be applied to monitor the metastasis and recurrence of the disease after operating on the patients, for the reason that both the sensitivity and the specificity of the tumormarker proteins, which are used for the tumor detection of digestive tract, are not high in screening of common people. Therfore, in clinic, it is clinically significant to look for new gastric cancer related molecular markers, including molecular markers in peripheral blood expecially in the world.The concept of Proteomics was first put forward by the Australian researcher,Williams in 1994, and the method was applied to scientific research for the first time.Its essence is the protein group as the research object.From the perspective of the whole, it analyzes protein composition of cell dynamically and its activity routines.The protein which is the final form of gene transcription not only takes part in the maintenance of life, but also adjusts concrete form of life activities. Research into the proteins and its continual perfection contributes to the early diagnosis of the disease as well as the research into the molecular targets and drugs used to treat the disease.The study uesed the method to research the serum of gastric cancer patients in order to find out the special protein markers for the purpose of diagnosing gastric cancer earlier. The proteomics technology was used in to clarify the relationship between the development of gastric cancer and specific serum markers, which could assist to explore a new way to treat gastric cancer.This study mainly tested the serums(preoperative group, postoperative group,normal serums group) of gastric cancer patients. By comparing the three serum groups of gastric patients with normal serums using proteomics SELDI TOF MS technology, this experiment aims to find out the specific proteins in gastric cancer patients serum and construct the differentially expressed proteins spectra. The target proteins were separated and purified by TRICINE-SDS-PAGE. Using MALDI-TOF/TOF technique tested and verified the potential target proteins. At last, the specific protein markers in serum of gastric cancer patients were confirmed. Whether it has an effect on the growth of cell is testified by cell culture and animal experiment,which may provide possibility and accordance for the diagnosis and treatment of gastric cancer.2. Objective Proteomics technology is applied to screen and identify the specific protein markers in the serum of gastric cancer patients, Cell culture and animal experiment are used to clarified whether the specific protein markers affect the growth of cancer cells.3.1 clinical and experimental Material The study collects 60 cases of gastric cancer which are first diagnosed in the general surgery of the tumor hospital affiliated to Zhengzhou University. The 60 cases include 46 male and14 female patients whose ages range from 42 to 84 and the average age is 58±0.5. 20 cases are proved to having metastasized when on diag nosis without surgical treatment are setted as metastasizing group by taking serum samples.The other 40 unequivocal diagnosed cases are setted as pre-operative group which takes serum sample the day before surgery and post- operative group which takes serum sample the 12 th day after surgery. All the cases haven’t accepted chemotherapy and radiotherapy yet before taking samples. All cases had been verified to be adenocarcinoma by surgical and pathological diagnosis, of which, 14 are highly differentiated, 21 are moderately differentiated, 25 are poorly differentiated. Serum of 20 cases was provided by chronic atrophic gastritis patients, 40 cases by healthy volunteers. All blood samples provided by 120 patients were extracted in the early morning on an empty stomach condition,The pathological diagnosis is all confirmed by more than two pathologist. The samples are all taken from fasting blood from the morning, and then placed in dry test tubes for natural blood coagulation at room temperature for 1 hour, then centrifuged at the speed of 3000 r/min for 15 minutes.And then the centrifugal supernatant after precipitation is extracted to the PE pipe and placed in the freezer to save at- 80 ℃. The study has passed the ethical review of the ethics committee in our hospital, and all the checked patients have affixed to the informed consent.The MGC-803 and HGC-27 cell lines used in the experiment were supplied by Shanghai cell bank of Chinese academy of medical sciences. BLAB/C nude mice were bought from Beijing Weitonglihua animal technology company. The mice were at the age of 4-5 weeks including 20 females and 20 males with weight of 12-15 g.3.2 The main reagents and instruments The protein biochip system(Ciphergen PBS II+ SELDI-TOF-MS)and protein chip WCX2 used in detecting target protein were purchased from American Ciphergen Biosystem company. The related reagent was bought from American3. Material and MethodsSigma company. Matrix assisted laser ionization time of flight mass spectrometry(MALDI–TOF–MS) was purchased from English Kratos Analytical company. Apo CⅠ was bought from China’s jersey brook source biological technology company.Fluorescence microscope is the product of Japan Olympus company. Vacuum cooling centrifuge enrichment system(SPD Speed Vac) was bought from American Thermo Electron company. 1640 medium and DMEM medium were the products of Israel BI company, and Mc Coy 5A medium and fetal bovine serum were purchased from the US GIBCO company. Trypsin was the product of solarbio company in China.Fluorescence microscope was purchased from Olympus company in Japan.High-speed centrifuge with super speed and low temperature was bought from Japan HITACHI company. The Mini- PROTEAN Tetra Electrophoresis System and Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell bought from the Bio-Rad Laboratories company in the United States. Small Series360 IBM servers(S/N: 99wlwx4) bought from the IBM company in the United States. Antiserum TTHY,antiserum Apo C-III, the rat resistance serum, the rabbit resistance serum bought from the santa cruz biotechnology company. PVDF membrane bought from the Millipore Corporation company in the United States.3.3 Experimental Methods The experiment methods are based on three procedures:3.3.1 The pretreatment of the samples and preliminary screening of characteristic protein Select 40 cases of gastric cancer patients and 20 cases of healthy volunteers as a training group randomly, aiming to filter characteristics protein; 20 more patients with gastric cancer, another 20 cases with chronic atrophic gastritis patients and 20 cases of healthy volunteers as the test group, in order to verify the validity of the target.Unfreeze the frozen serum we have collected in ice water, then extract the supernatant after centifugal again, then place the supernatant on well-processed protein chip. Next, dry the chip, fix it in bioprocessor and keep a record, waiting for the test of computer. Select standardized correction program to correct the system:use protein chip with known molecular weight to correct the SELDI-TOF-MS system to the error less than 0.1%. Regulate the other related parameters of the mass spectrometer, such as laser intensity and data sampling sensitivity to the best state.1000Da-30000 Da is set as the scope of data collection of protein detection, and2000Da-20000 Da is the best range. All equipments prepared, we place the protein chip on the target of the mass spectrometer for detection, and collecting the experimental data in computer. Analyzing the experimental data with the software,and calculating the m/z peak of different protein in each sample, when the m/z peak is less than 0.3%, the two proteins are considered to be the same kind. m/z peak data of different experimental groups is analyzed by Wilcoxon rank sum test in computer.Differential analysis is on basis of the size of P values, the smaller P value means the greater difference in the intensity of expression, and also the greater clinical significance, and then we find out discrepant protein peak in different groups. Choose the inspection standard as a = 0.01. Using the nonlinear support vector machine(SVM) classifier to analyze the essential spectrum data of every sample, then establish the discriminant model, verify the validity of the established model using cross validation method.3.3.2 Further separation and refinement of differential proteins The differential proteins, after being found, were separated through gel electrophoresis. Firstly, the colloid was prepared which was used for gel matrix.Secondly, the processed serum specimens were separated through SDS-PAGE. In accordance with the molecular weight of target proteins, the corresponding parts would be cut off from the separated gels on the different axis of molecular weight and then be put into different EP tubes respectively. Thirdly, in accordance with the kit’s instructions, the enzymic hydrolysates would be the target proteins after washing,bleaching, drying and adding trypsin. The next, we dripped the extraction liquor and then collected the supernatant which was arrayed onto protein chips after other processing. Finally, we identified them by MALDI-TOF-TOF to get eligible peptide fragments.3.3.3 Validation of the markers of serum protein for gastric carcinoma After the serum was dissolved and centrifuged, collect 100 ul supernatant fluid.Use 103 v constant voltage into the protein gel electrophoresis. Using the electrophoresis bromophenol blue to get the gel as the finish. Separate the electrophoresis gel and electrophoresis plate, cutting out the corresponding protein band according to the internal reference, adopt the three layers of filter paper : PVDF membrane- gel- three layers of filter paper in a way that overlay, followed by a constant 20 v voltage to transfer membrane. Obtain the target protein by immune response and chemiluminescence and photographic fixing technology. Scanning the negative film, assay each protein negative gray scale of every image using image pro plus 6.0 software. Use SPSS, version13.0 statistical software to analyze the statistics difference of the gray value in groups.3.3.4 Influence of target protein on proliferation of gastric cancer cell lines The gastric cancer cell lines were digested, re-elected, and counted. Lay aside 96- well plates, about 3500 cells per hole, 4 holes per group, a total of 6 groups, 100 ul medium per hole, 37℃ cultivation overnight, completely sticking wall, growing well,proliferation in logarithmic phase. Configuring different density of peptides,dissolving peptides by medium, extracting supernatant after centrifugation, divided into 5 levels: 0.1, 0.5, 1.0, 1.5 and 2.0ug/ul. Usting another medium without peptides as control group, adding different concentrations of peptides into the culture medium of the other five groups, besieds, setting another group, 100 ul medium without cells.After the first 24 h after the start, adding 10 ul CCK-8 reagent to each hole every 24 h,hatching in1.5 h, detecting its absorbance to 72 h according to 450 nm of light absorbance value.3.3.5 Influence of target protein on tumors of tumor-burdened mice Choose BLAB/C 20 male and 20 female nude mice which weigh 12-15 g. Using the MGC-803 cell lines, we fully extended the amplification and recount after digestion of the exponential phase cells to guarantee that 5 x106 cells(0.2 ml) could be injected to each mouse. The back skin of mice was injected with 0.2 ml cell suspention liquid.The mice were fed in SPF animal center. Observe the growth of tumor regularly and measure the mice weight and size of tumor. These mice were dosed in the second week separately. 10 mg peptides were dissolved in 1ml normal saline to achieve the density of 10mg/ml. The dose injected to the mice through caudal vein is 100mg/Kg. Injecting the mice for 5 days and observing their vital signs,tumor size and the weight. Mice were euthanized after 1 week. Tumors were collected,weighed and measured. At last, the inhibition rate wass calculated. The expression level of Ki67,Bcl-2 and Bax were tested by immunohistochemistry.4. Results4.1 By analyzing the protein spectrum number of the 60 cases, the m/z peak of all the sample proteins, which are regarded as the same kind if the difference of peak is no bigger than 0.3%. Through the Wilcoxonrank sum test on the two groups’ peak,15 specific m/z peaks were acquired(P<0.01), including 8 lower expression peaks and 7 higher expression peaks in preoperative gastric cancer group. Making use of the SMV to screen out composite pattern with highest youden index, we got a protein whose m/z peak was 6438. It highly expressed in normal group whereas it expressed low obviously in preoperative gastric cancer group. The difference of two groups are statistically significant(P<0.01). Its expression level was much higher in postoperative group, while the expression level was lower in preoperative group.Referencing to composite pattern with highest youden index, the m/z peak of the protein we got was 6438. It expressed much lower in metastasis group than preoperative group. The difference of two groups are statistically significant(P<0.01).4.2 The peptide E.LK EFGNTLEDKA RELISRIKQS ELSAKMREWF SETFQKVKEK.G was detected from purified liquid with m/z peak of 6438. Using Mascot searching software, we connected Swiss Prot database and the protein was identified as a peptide of Apolipoprotein CⅠ(apolipoprotein C-I, Apo C-I).4.3 The verification result of the otherness protein markers of gastric cancer’s serum.We measured the serum Apo C- Ⅰ, TTHY and Apo C-III of preoperative,postoperative group and normal control group by Western blot to statistics-analyze the grey value of the target protein. As a result, the gray difference of the preoperative group and the normal group ‘s protein was statistically significant(P < 0.05), as well as the gray difference of the preoperative and the postoperative group’s protein(P <0.05).4.4 The analogs Apo C-Ⅰ apparently the growth of gastric cancer lines of HGC27, MGC803 and the effect was positively related with the density and times of dose.4.5 Apo C-Ⅰhas an negative effect on the growth of tumors of mice injected with MGC803. Compared with the 0.8+0.1g weight of the control group, the weight of experimental group was 1.2+0.2g, with 33.3% inhibition ratio and apparent statistic differences(P<0.05). The expression level of prolification indicators: PCNA,Ki67 and Bcl-2 was lower while the expression level of apoptosis indicator: BAX was higher.5. Conclusions5.1 The protein with the m/z peak of 6438、14222 and 8622 is identified as Apo C-I、TTHY and Apo C-III, which would direct the further exploration of serum specific protein markers in gastric cancer. The expression level of protein with the m/z peak of 6438 has an obvious relationship with the tumor progression in gastric cancer, which offers new thoughts for later studies of the correlation between specific protein markers and the development of gastric cancer.5.2 We identified Apo C-I 、 TTHY and Apo C-III as potential proteomic biomarkers of PTC.5.3 Analogs Apo C-I peptide has a lethal effect on gastric cancer lines and tumors of mice, which can provide the theory foundation for later research into the relationship of them..5.4 Analogs Apo C-I may become a new kind of protein anti-tumor drug and new targeted drug for gastric cancer treatment, which may also play an important role in the treatment of other types of tumors.