The Identification And Study Of Human Pancreatic Cancer Associated Immunogenic Antigens SLP-2 And TOM40

BACKGROUNDThe human pancreatic cancer is one of the most deadly malignancies with silence onset,rapid progress and poor prognosis.Early diagnosis of pancreatic cancer plays a central role in the comprehensive therapy system which combined the surgery and other therapies such as chemotherapy and radiotherapy.The screening of serum tumor marker was considered to be one of the most hopeful methods to make a progress in pancreatic diagnosis research.Our lab presented a new concept to detect pancreatic cancer which planed to use the antibodies of immunogenic associated membrane antigens,and also presented the concept of pancreatic cancer associated immunogenic membrane antigens and designed a new screening strategy for the pancreatic cancer associated immunogenic membrane antigens.After the previous work of our research team which were based on the pancreatic cancer cell line extracted membrane proteins,Western blot and proteomics,the IgG of SLP-2 and TOM40 were detected in the serum of pancreatic cancer patient without in the normal persons and chronic pancreatitis patients,so the antibodies of SLP-2 and TOM40 were considered to have potential diagnosis value in pancreatic cancer,and the expression condition of SLP-2 and TOM40 in pancreatic cancer are needed to be further investigated.OBJECTIVETo study the expression of SLP-2 and TOM40 in human pancreatic cancer both in the transcript level and translation level,and to identify the SLP-2 and TOM 40 as pancreatic cancer associated immunogenic membranes,and also conduct some initial study of these two proteins which are associated with pancreatic cancer and do some basic work for the future investigation of the 2 proteins in pancreatic cancers.METHODSThe Real time PCR,Western blot and immunohistochemistry of human pancreatic cancer tissue microarray were used to detect the expression of SLP-2 and TOM40 in different levels of human pancreatic cancer.Bioinformatics analysis was conducted to both gene and protein of SLP-2 and TOM40 by the online database such as NCBI, ASTD,Oncomine Research Platform,Gene Ontology,UCSC Genome Browser,and also the local bioinformatics software such as ESyPred3D,ClustalX,and TreeView.etc.The analysis included the evolution of protein,the gene alternative splicing and transcript, electronic expression spectrum,the prediction included miRNA and the antigen epitope of B cell and cytotoxic T lymphatic cell.The immunologic fluorescence cytochemistry method was conducted to the pancreatic cell line AsPC-1,BxPC-3,PANC-1 and the confocal laser scanning microscopy was used to identify the position of the SLP-2 and TOM40 and also the related expression levels in the 3 pancreatic cell lines.The vector pGPU6/GFP/Neo was used to construct the SLP-2 RNA interfering vector.RESULTS1.Real time PCR detected the expression of SLP-2 and TOM 40 in both 10 cases human pancreatic caner and 10 cases normal human pancreatic tissue,and the TOM40 had high expression level in pancreatic cancer tissue compared to the normal pancreatic tissue.2.Western blot detected the expression of SLP-2 and TOM40 in both 10 cases human pancreatic caner and 10 cases normal human pancreatic tissue;the immunohistochemistry of human pancreatic tissue microarray indicated the expression of SLP-2 and TOM40 in pancreatic cancer,the SLP-2 and TOM40 had high expression levels in pancreatic cancer tissues compared to the normal pancreatic tissues.3.The protein evolution trees of SLP-2 and TOM40 was constructed,and the transcript factor prediction analysis indicated that the SLP-2 might be regulated by CEBP and ELK1 ect,and the TOM40 might be regulated by the CDPCR3,PAX5 etc.The miRNA prediction presented the miRNA which might regulated the SLP-2 and TOM40,and the prediction 3-D structure of SLP-2 was made,and the antigen epitope of B cell and cytotoxic T lymphatic cell were predicted and presented.4.The confocal laser scanning microscopy observation of pancreatic immunologic fluorescence cytochemistry indicated that both SLP-2 and TOM40 was expressed in the cytoplasm of pancreatic cancer AsPC-1,BxPC-3 and PANC-1.The PANC-1 cell line had the highest expression level of both SLP-2 and TOM40,and the AsPC-1 cell line had the lowest expression level.5.The SLP-2 RNA interfering vector pGPU6-GFP-Neo-SLP-2-shRNA was constructed and the pancreatic cell line SW1990 was transfected successful.CONCLUSIONS1.SLP-2 and TOM40 were identified as the human pancreatic cancer associated immunogenic membrane antigens.2.The SLP-2 and TOM40 proteins mainly localized in the cytoplasm of pancreatic cell line AsPC-1,BxPC-3 andPANC-1,the expression of both SLP-2 and TOM40 had significant difference in these three pancreatic cell line,the PANC-1 had the highest expression level but the AsPC-1 had the lowest one.