| BackgroundBreast cancer has been one of the most common types of malignant tumor in female all over the world, there are 1.3 million people per year diagnosed with breast cancer, and about 4000,000 people per year died. The morbidity and mortality of breast cancer rank second in female tumors worldwide[1]. In China, the morbity has ranked first in female tumors, and increased by 3%~4% per year. The morbidity of breast cancer rises with the age, and the postmenopausal woman will catch a high risk of suffering from breast cancer. While there are 15%~25% of the females who were diagnosed with breast cancer are premenopausal. The 7% of which were below 40 years old. A considerable number of young women of childbearing age were diagnosed with breast cancer, so the patients’ s physical and mental health and the life quality were seriously threatened. The prognosis of breast cancer are associated with multiple factors,such as the histomorphological types,clinical staging,and whether exists distant metastasis. The breast cancer patients will suffer from a high risk of metastasis all her life. Metastasis and relapse have yet been the main obstacles of survival time of breast cancer patients. Recently,the reason of high mortality of cancer is the lack of biomarkers used to screen and diagnose cancer. If we can find apanel of biomarkers which can detect the breast cancer in the subclinical stage,then can help prevent or reverse the development of breast cancer by early interventions,and improve the prognosis to a great extent. Although the serum biomarkers have not yet played a major role in breast cancer diagnostic or prognostic practice[2], an effective biomarker panel in an easily accessible biological fluid would be a valuable and minimally invasive adjunct to other clinical and pathological approaches. So it’s of important significance to find a newprotein markers which can help to make an early diagnosis, make a correct prognosis and evaluate the curative effect[3].Proteomics can contact with and reflect the changes of oncogene and cellular physiology,so people place high hopes on finding tumor biomarkers via protein analysis technology.Matrix-assistedlaserdesorption/ionization,time-of-flight mass spectrometry(MALDI-TOF MS) and the derived surface-enhanced laser desorption/ ionization mass spectrometry(SELDI-TOF MS)enable the development of high-throughput proteome analysis based on comprehensive reliable biomarkers, and it also provide very large development space of the research of new panel of biomarkers in the serum of breast cancer. The protein profiles between tumor tissue and normal tissue, or the body fluid of tumor suffer and normal people can be compared via SELDI-TOF-MS, proteins of differential expression can be found, namely tumor associated proteins. Many researchers think that the accuracy can be improved if we can predict and diagnose disease according to the proteome changes. It’s more accurate and effective to detect the proteome changes than gene changes.This research analyze the serum of breast cancer and healthy females by surface-enhanced laser desorption ionization time-of-flight mass spectrometry(SELDI-TOF-MS),research the proteome profiles between the breast cancer preoperative group,postoperative group,relapsing group and normal people,and the proteome profiles between triple-negative breast cancer and non-triple negative breast cancer, to find the related serum biomarkers. ObjectiveThrough the proteomics to research the proteome profiles between the breast cancer preoperative group,postoperative group,relapsing group and normal people, and the proteome profiles between triple-negative breast cancer and non-triple negative breast cancer,to find the differential expresseing proteins, and finally to ensure a panel of biomarkers which can reflect the exists of tumor,evaluate the curative effect and identify different molecular types of tumor,and build a perfect protein fingerprint pattern model for early diagnosis,curative evaluation and prognosis analyzing. Materials and methods Materials:Collect the materials of 60 cases of female breast cancer patients without any treatment since January 2010 to Jane 2014.,aged 22 ~69 years old,mean age 44.6+0.3 years old. All of them received operation,and all diagnosis were confirmed by postoperative pathologic.The patients received regular radiotherapy or chemotherapy after operation,while these factors weren’t considered in the research. 22 cases out of 60 were triple-negative breast cancer patients,11 cases relapsed after operation,inside which 8 cases were triple-negative breast cancer.Collect 20 cases who were without breast disease as controls. The researchers had informed the patients of the content of the research, and had gained approval.Collect the serum of all the breast cancer patients before operation, 2 weeks after operation, both were 60 cases. Draw the blood specimens in the morning at 5 or 6 o’clock before eating, and using red head drying tube, placed the specimens at room temperature,0.5-1hours later centrifuged them at 3000 r/min for 20~30min. Extracted the serum and preserved in-800 C fridge in 1 hour. Methods:1)Dealing with breast cancer serum,unfreeze the serum specimens in the ice cake, centrifuging 3~5min after dissolution.Treated 96-hole protein chips are loaded into bioprocessor,and make detailed record, preparing for detection.2)Reset SELDI-TOF-MS system,use known proteinchips to adjust SELDI-TOF-MS system,to ensure that the molecular weight error were limited to 0.1%.Through editing operation program,Set the parameter of mass spectrometer, the best laser intensity and detection sensitivity.,set the highest molecular weight to 30000 Da, while the best scope is 2000Da-20000 Da.Select the primary screening result, and analyze the data through specific software, obtain the m/z peaks in each sample,it is often considered as the same class if the differences are less than 0.3%.m/z peaks in different groups were analyzed by Wilcoxon rank sum test.We consider it has significant difference if P<0.01.So we can get different m/z peaks between tumor group and the control group,or between different types of tumors,then find out approximate value ranging about +0.3%. 3) Seperation and purification of the serum by SDS-PAGE, seperate proteins of different molecular weight in serum through gel electrophoresis, and cut the targeted stripes, according to the specifiactions of enzymatic agent, after washing,decoloration and drying, add trypsin to get the target protein.4)The identification of target protein,add in extracting solution, centrifuged at 10000r/min for 10-15 minutes,then collect the supernatant.Spotting the sample on the chip,then put it into MALDI-TOF-TOF,collect the peptide fragments, then Connect Mascot software and Swiss Prot database,finally get target protein. Results 1 Normal control group,preoperative group, postoperative group and relapsing groupThe original data elimina through Wilcoxon rank sum test.3 peaks with high expression and 1 peak with low expression in preoperative group. 3 m/z peaks of high expression were 6683.8 Da、4695.0 Da、' 3983.8 Da.They all have a high expression in preoperative group, the expression decreased in postoperative group while increased in relapsing group. Through SVM to screen out models with maximum youden,m/z 6683.8 Da was obtained.High expression in preoperative group,the intensity is 898.9+55.9,while has a low expression in the control group,the intensity is 118.6+37.5.the intensity in postoperative group is 194.7+44.1,while the intensity in relapsing group is 474.2+83.2. The results between normal group and preoperative group, preoperative group and postoperative group,postoperative group and relapsing group were all contracted obviously,with significant difference( P<0.01).While there are no significant difference in normal group and postoperative group,preoperative group and relapsing group(P>0.01). 2 Triple-negative group and non-triple negative groupUsing SELDI-TOF-MAS to obtain specific peaks between Triple-negative group and non-triple negative group,From comparison to get approximate peaks ranging about +0.3%.We obtain 8 pecific peaks(P<0.01),4 peaks with high expression in triple-negative group and 4 peaks with high expression in non-triple negative group.Through SVM to screen out models with maximum youden,we also obtained m/z 6683.8.The intensity in triple-negative is 805.4+65.7,and the intensity in non-triple negative group is 206.3+29.0. The results have statistics meaning(P=0.001637<0.01). 3 Identification of specific protein peaksAfter the separation,purification and enzymolysis of target protein in the serum of breast cancer, using MALDI-TOF-TOF to detect peptide fragment mixture,m/z 6683.8 Da was identified as apo C-III. Conclusion1.m/z 6683.8 Da peak was identified as apo C-III,it can be considered as a specific kind of serum biomarker of breast cancer.2.m/z 6683.8 Da peakcan help distinguish triple-negative breast cancer from non-triple negative breast cancer. |
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