Identification And Functional Study Of Novel Protein Markers In Colorectal Cancer By Protein Microarray

Colorectal cancer(CRC)is one of the most common and most important cancers.and it is the second leading causes of cancer-related deaths in the world.The prognosis of CRC is dependent upon the extent of the disease.As colorectal cancer patients are often asymptomatic in early stage,that most colorectal cancer patients are diagnosed in late stage,missed the best treatment time,seriously affect the treatment and prognosis.Approximately 60%of patients who undergo curative resection experience local recurrence or distant metastasis.Survival rates of patients with CRC have increased in the past few years,possibly as a result of early diagnosis,changed patterns in colorectal cancer risk factors and improved treatments.The incidence of CRC is closely related to the lifestyle and dietary structure.As people life style,diet structure changes,life expectancy increases,the incidence of CRC will also further improve,and there are more younger patients.Therefore,it is very important for early colorectal cancer screening.At present,options for CRC screening include high-sensitivity fecal occult blood testing and colorectal endoscopy.Fecal occult blood testing is the most widely used means of screening for CRC,because of its ease of performance,lower cost and painless.But fecal occult blood testing has poor specificity and sensitivity.In many other diseases,such as peptic ulcer,upper gastrointestinal hemorrhage,the result of fecal occult blood testing may be positive.And only when the amount of bleeding reach certain amount fecal occult blood can be detected,that most fecal occult blood positive patients are advanced colorectal cancer.Colonoscopy is the most effective method for colorectal cancer screening,and it is the gold standard for colorectal cancer diagnosis.In the United States,thanks to colonoscopy,the incidence of CRC of adults aged 50 years and older decreased significantly.But colonoscopy preparation is troublesome,expensive,painful,as a result about a third of the patients give up colonoscopy examination due to poor tolerance.And someone who suffers from a serious cardiovascular disease and coagulopathy can't take colonoscopy examination.So colonoscopy examination can not be directly carried out in asymptomatic populationIn general,serum tumor markers can be an easy-to-use approach for the detection of cancer,carcinoembryonic antigen(CEA)and carbohydrate antigen 199(CA199)are the markers most frequently evaluated as a predictive biomarkers in patients with CRC.However,CEA and CA199 levels in many other tumors are increased,and they are meaningless in the early diagnosis of colorectal cancer.Therefore,how to choose high sensitivity,high specificity serum tumor markers of CRC have greater significance.Proteomics is an effective method for the identification of new tumor markers or the key proteins in tumor development and progression.Antibody microarray is an effective tool for proteomics research.In this study,the author used human cytokine antibody array and to detect the expression of 274 cytokines in the serum of patients with colorectal cancer and normal control group.And then use enzyme linked immunosorbent assay(ELISA)for verification in more patients,thereby providing markers CEACAM1,Cripto-1,and VCAM-1 41BB for early colorectal cancer screening.At last the author used immunohistochemical method to further analyze their expression in colorectal cancer tissues and the relationship between the clinical features to explore the means in occurrence,development,prognosis,treatment and curative effect.Objective1.To detect the differentially expressed proteins in the serum of colorectal cancer patients,and to screen out the high expression proteins in the serum of patients with colorectal cancer,to find new tumor markers for early screening of colorectal cancer.2.To detect the expression of differentially expressed proteins in colorectal cancer tissues and their corresponding normal intestinal mucosa tissues,and to analyze the relationship between the expression and the clinicopathological parameters,to explore the significance of these proteins in occurrence,development,treatment and prognosis of colorectal cancer.Materials and methods1.Serum of 5 patients with colorectal cancer preoperative and normal control group were collected.274 cytokines were simultaneously analyzed using the human cytokine antibody array(RayBio)to choose highly expressed proteins in patients with colorectal cancer.2.Choose the highly expressed serum proteins in patients with colorectal cancer,ELISA and statistical analysis was applied in 30 cases of colorectal cancer patients,to analysis the significance in the early diagnosis of colorectal carcinoma.3.The expression of highly expressed proteins were detected using immunohistochemical staining in colorectal carcinoma tissues.The relationships between the proteins and clinicopathological parameters were analyzed to explore the significance of the proteins in occurrence,development,treatment and prognosis of colorectal cancer.Rusults1.274 cytokines were simultaneously analyzed using the human cytokine antibody array AAH-CYT-G4000.P values less than 0.05 were considered statistically significant.In addition,fold change(FC)was calculated.The FC values of cytokines are given to indicate their relative expression levels.Any FC greater than or equal to 1.5 or less than or equal to 1/1.5 in signal intensity among the groups was considered relevant.The results showed that 94 proteins were differentially expressed,among which there were 66 proteins significant highly expression in the sera of colorectal cancer patients,they are AFP?IL-17 R?Cripto-1?CA199?CALCA?DKK-4?TG?CEACAM1?CEA?4-1BB?HER2/ErbB2?CDH1?IL-17F?Ubiquitin?NGF R?ANGPTL4?Angiostatin?CCL14?TGF beta2?CA9?BMP-5?CTSS?GDF-15?FSH?CD23?S-100b?TRAIL R2?VCAM-1?CA125?DKK-1?IP-10?beta 2 M?MDC?DR6(TNFRSF21)?Fas Ligand?LIF?Shh N?IL-2 Rbeta?TIMP-4?CRP?Adiposin?sgp130?Hcg intact?HVEM?CA15-3?BMP-7?IL-13R alphal?LYVE-1?MMP-9?TNF-beta?IL-9?NAP-2?MIP-ldelta?ErbB3?PSA-free?IL-6?IL-18Bpalpha?CD40 Ligand?NIDI?IL-3?IL-lalpha?CXCL-16?Angiogenin?ALCAM?FSTN?BCAM?As well as there are 28 proteins significant lowly expressed in the sera of colorectal cancer patients?they are IGFBP-2?VEGF R3?Insulin?Resistin?PDGF Rbeta?VEGF R2?TREM-1?SCF R?TIMP-2?CD30?PDGF-BB?ICAM-2?Osteoprotegerin?PSA-total?TSH-??Upar?Stnf R??EGF-R?MMP-3?L-Selectin?Sil-6 R?MICB?E-Selectin?Axl?HCC-4?TRAIL R3?Stnf-RI?MSP-alpha?2.ELISA was used to verify the differentially expressed proteins,results showed that CEACAM1,Cripto-1,VCAM-1 and 41BB levels were significantly higher in sera of colorectal cancer than the normal control group,statistical analysis of ROC curve showed that CEACAM1,Cripto-1,VCAM-1 and 41BB has good significance in early prediction of colorectal cancer with high sensitivity and specificity.3.Immunohistochemical staining were used to analysis the expression of CEACAM1,Cripto-1,VCAM-1 and 41BB in colorectal carcinoma tissues.The results showed that the expression of CEACAM1,Cripto-1,VCAM-1 and 41BB were significantly higher in colorectal cancer tissues.High expression of CEACAM1,Cripto-1,VCAM-1 and 41BB was correlated with clinical stage,and high expression of CEACAM1,Cripto-1 and VCAM-1 were highly correlated with lymph node metastasis.Conclusions1.The protein array can simultaneously and efficiently detect the expression of various proteins in the tumor,which is a good method for screening tumor markers.2.CEACAM1,Cripto-1,VCAM-1 and 41BB were highly expressed in serum of patients with colorectal cancer,serum CEACAM1,Cripto-1,VCAM-1 and 41BB level is helpful in the early screening for patients with colorectal cancer,and it is non-invasive.3.CEACAM1,Cripto-1 VCAM-1 and 41BB are highly expressed in colorectal cancer tissues,is helpful in judging the prognosis of colorectal carcinoma,and may become a new target for the treatment of colorectal cancer patients.The innovations of this study1.Detect the expression of 274 cytokines in serum of patients with colorectal cancer and normal controls at the same time,and provide new serum tumor markers for colorectal cancer screening.Then detect the tumor makers in serum of patients with colorectal cancer by ELISA,and do statistics analysis to verify the tumor markers,select new colorectal cancer tumor markers CEACAM1,Cripto-1,VCAM-1 and 41BB,and provide experimental basis for the early diagnosis of colorectal cancer in clinical.2.The expression of the new protein markers were detected by immunohistochemical staining in colorectal carcinoma tissues,and analyze there relationship with clinical characteristics of colorectal cancer,to explore their roles in occurrence,development,curative effect and prognosis in colorectal cancer.