The Interaction Of MT And Zinc Alpha2Glycoprotein In Esophageal Cancer

Objective: Esophageal cancer occur in esophageal epithelial cell ofmalignant tumor,clinical also known as esophageal cancer, accounting forabout2%of all malignant tumors. Accounted for22.34%in malignant tumordeath toll in the country, is second only to stomach cancer, accounting forsecond place. In China, esophageal cancer found in men in the age of40, theincidence of the esophageal cancer in general is the ratio of men more thanwomen, men and women was1.3-2.7:1, but in recent years under the age of40,growth trend of disease, and has obvious characteristics of the racialdifferences. Now about esophageal cancer tissue and normal tissue betweenproteomics study reported less,search and select specific differential proteinsassociated with esophageal cancer in the interaction between is of greatsignificance to evaluate the occurrence of esophageal cancer.This study intendto use co-immuneprecipitation(co-immunprecipitation, Co-IP) and SDSamido-polypropylene gel electrophoresis (SDS-Polyacrylamide gelelectrophoresis sds-page, SDS-PAGE) technology combined with Liquidphase mass spectrometry, Liquid chromatography-mass spectrometry, LC-MS)technology, in normal esophageal tissue and esophageal cancer tissue proteinscreening, to find the differences between MT and zinc-alpha-2-glycoproteinwhich is interacting with MT protein, in order to find a can reflect is closelyrelated to the tumor protein and interacting proteins, for esophageal cancerdifferentiation mechanism and clinical research to provide theoretical andexperimental basis, and by intervening in some genes or signaling pathways(difference) to inhibit tumor cells and achieve the goal of treatment.Methods:1We collect the Normal esophageal tissue and esophageal cancer, selectsixteen cases of esophageal cancer tissues by pathological diagnosis. There were10males and6females,with the age between42to76years old. Thepatients didn’t do any treatment, chemotherapy and anything else beforesurgery. Among themThe numbers in the upper,middle,and lower part of theesophagus were2,8,and6.There were2stageⅠcases,5stage IIA cases,4stageIIB cases,4stageⅢcases,and1stageⅣcase.There were eight cases in welldifferentiated carcinoma,54cases in moderately differentiated carcinoma,and6cases in poorly differentiated carcinoma. We collected the totle solubleproteins and tested the concentration of protein samples using the Bradfordmethod.2We separate the proteins by co-immunprecipitation,with1μg antibodies(rabbit anti MT polyclonal antibody) adding in120μg sample from eachgroup.After that we mild shake them overnight under4℃, and then extractMT and its associated protein complexes;3Use SDS-PAGE to separate the proteins, then compare the gelelectrophoresis of the Normal esophageal tissue and esophageal cancer tissuesso as to find and excise the different protein bands.4We dissolve the different protein bands filtered in the gel, analyze thedifferentially expressed proteins extracted by LC-MS and then identify theprotein by biological information database.5We select and verify three of the eight differentially expressed proteinswhich were identified by Western blot on protein expression levels in order totest whether it maintains consistency with the proteomics test results.6Statistical analysis: Use Fujifilm Las-4000Statistical analysis softwareto analysis integral optical density in the band which we get, with β-actin asinternal reference. Then use software SPSS13.0statistics processing line andsingle factor analysis of variance between groups. All the test results recordedaccording to the mean and standard deviation（x±SD）, and EXCEL mapping.Results:1We get the gel map of normal esophageal tissue and esophageal cancertissue by Co-IP and SDS-PAGE. We get64different protein bands bycomparison, then identify31proteins by LC-MS.BY screening the high abundance and structural protein, we determine three proteins that may berelated to esophageal tumor. They are Lysozyme C, zinc-alpha-2-glycoproteinand lipocalin-1isoform1.2Then we verify zinc-alpha-2-glycoprotein in the protein levels. Theresult of western blot shows that MT strip containszinc-alpha-2-glycoprotein,and zinc-alpha-2-glycoprotein are significantlyenhanced, compared with the normal esophageal tissue（t=5.139， P<0.01）,which is also consistent with the proteomic result.Conclusions:1We find MT and many proteins play a role together in esophagealcancer tissues and normal esophageal tissues. It confirms that thezinc-alpha-2-glycoprotein and MT for interacting proteins. They togetherinvolve in tumor occurrence and development process, as well as tumor celldifferentiation, proliferation, and apoptosis.2Zinc-alpha-2-glycoprotein is verified by Western blot on the proteinexpression levels. The results are consistent with the proteomics results, andthis confirms that the protein expressions are different in esophageal cancertissues and normal esophageal tissues.3We build the map of esophageal cancer tissues and normal esophagealtissues, and identify three differentially expressed proteins. We find out theclosely related proteins with tumor differentiation, which provide theoreticaland experimental basis for tumor gastric cancer differentiation mechanism andstudy of reversal.