Discovery And Verification Of Colorectal Cancer Tumor Markers ANXA4?PLOD3 And SERPINH1 Based On Proteomics

Colorectal cancer(CRC)is one of the common malignant tumors of digestive tract.CRC is reported to account for approximately 10% of cancer and its related deaths worldwide,and CRC is the second most common cancer in women and the third most common cancer in men.Early diagnosis and treatment can significantly improve the survival rate of CRC patients,but the known tumor markers generally have low sensitivity and cannot meet the needs of early tumor prediction.It is an urgent problem to find CRC tumor markers with high sensitivity.Objective:The purpose of this study is to apply proteomics technology to analyze tumor tissue and normal tissue of CRC patients to identify and filter some differentially expressed proteins,and to select potential tumor markers of CRC for further verification and to conduct the clinical large sample experiments in order to judge its clinical application value.Methods:1.The shotgun proteomics technology based on nano-LC-MS/MS was used for proteomic analysis of 71 cases of CRC pathological specimens.The Uniprot database was used to search,identify and select differentially expressed proteins.2.Bioinformatics analyses such as GO,KEGG,PPI were performed for differentially expressed proteins in CRC.3.Western blot was used to verify the expression of some differential proteins in CRC tissues.4.Through the combined analysis of TCGA database and CPTAC database,ANXA4,PLOD3 and SERPINH1 in GO:0062023(collagen-containing extracellular matrix)were selected as potential tumor markers for CRC for follow-up research.5.RT-qPCR was used to detect the mRNA levels of ANXA4,PLOD3 and SERPINH1 in CRC tissues.6.HPA database and immunohistochemical experiment(IHC)were used to detect the expression and subcellular localization of ANXA4,PLOD3 and SERPINH1 in CRC tissues,and the correlation between the expression levels of the above proteins and clinicopathological parameters was statistically analyzed.7.Western blot and RT-qPCR were used to detect the protein and mRNA expression of ANXA4,PLOD3 and SERPINH1 in CRC cell lines.8.ELISA method was used to detect the expression levels of autoantibodies specific for ANXA4,PLOD3 and SERPINH1 in the peripheral blood of 260 CRC patients and 100 healthy people in large-scale clinical experiments to determine their clinical application value.Results:1.In the proteomic study of CRC,a total of 2969 proteins were identified.According to the principles of expression fold change and significant difference analysis,43 differentially expressed proteins of CRC were selected including PLOD3,S100A11,SERPINH1,HUWE1,ANXA4,PADI2,CTSL,CTSZ,PTPN6,etc.2.The results of bioinformatics analysis showed that in terms of GO-CC,most of the differentially expressed proteins were located in Golgi vesicles,transport vesicles,coated vesicles,extracellular matrix containing collagen,etc;in terms of GO-MF,the differentially expressed proteins were mainly in binding function and catalytic activity,with phospholipid binding,proteoglycan binding,amide binding,sulfur compound binding,peptide binding,etc;in terms of GO-BP,differentially expressed proteins are mainly involved in antigen processing,presentation of exogenous peptide antigens,neutrophil degranulation,activation,Golgi vesicle transport,etc.KEGG analysis showed that differentially expressed proteins were involved in phagosome,lysosome,antigen processing and presentation,protein processing in endoplasmic reticulum,cell adhesion,apoptosis,etc.The results of PPI analysis showed that the interactions between the differentially expressed proteins were relatively close,forming a complex protein interaction network.There were key nodes among the differentially expressed proteins including HLA-DQA1,HLA-DRB1,SEC24 C,and SEC24 D.3.Western blot results showed that compared with normal tissue,there were significant differences in the expression of up-regulated proteins SERPINB5,SERPINH1,PLOD3,ANXA4 and down-regulated proteins PADI2 and CTSZ in tumor tissue of CRC(P<0.05),which verified the reliability of proteomic analysis results.4.Through the combined analysis of TCGA database and CPTAC database,ANXA4,PLOD3 and SERPINH1 in GO:0062023(collagen-containing extracellular matrix)were selected as potential tumor markers of CRC for follow-up research.5.RT-qPCR results showed that compared with normal tissue,the mRNA expressions of ANXA4,PLOD3 and SERPINH1 were significantly increased in tumor tissue of CRC(P<0.05).6.The results of HPA database and IHC assay showed that compared with normal intestinal epithelial cells,the expression of ANXA4,PLOD3 and SERPINH1 in the cytoplasm of CRC tumor cell was obviously increased.7.The expression of ANXA4 and SERPINH1 protein was significantly correlated with TNM stage and lymph node metastasis(P<0.05).The expression of PLOD3 protein was significantly correlated with pathological type,TNM stage and lymph node metastasis(P<0.05).8.Compared with NCM460,the mRNA expressions of ANXA4,PLOD3 and SERPINH1 were significantly increased in most CRC cell lines such as Lo Vo and HCT116(P<0.05).9.Compared with NCM460,the protein expressions of ANXA4,PLOD3 and SERPINH1 were significantly increased in most CRC cell lines such as SW620 and HCT116(P<0.05).10.ELISA results showed that: 1)The expression level of ANXA4 autoantibody in the Stage I+II group was significantly higher than that in the Control group(P<0.0001).ANXA4 antibody was significantly higher in the Stage III+IV group than in the Stage I+II group(P=0.0349).The expression of the Chemotherapy group was significantly lower than that of the Control group(P=0.0126).2)The expression level of PLOD3 autoantibody in the Stage I+II group was significantly higher than that in the Control group(P<0.0001).3)The expression level of SERPINH1 autoantibody in the Stage I+II group was lower than that in the Control group,but the difference was not statistically significant(P=0.3552).Conclusions:1.The differentially expressed protein profile in tumor tissue and normal tissue of CRC was obtained by proteomics research in this study.2.ANXA4,PLOD3 and SERPINH1 was significantly differentially expressed in CRC,and can be used as candidate tumor markers for CRC.3.The expression of ANXA4 and PLOD3 autoantibodies were significantly increased in the Stage I+II of CRC,and can be used as candidate early tumor markers for CRC.4.The level of ANXA4 autoantibody was significantly increased with the progression of tumor stage(from Stage I+II to Stage III+IV),which can reflect the progression of CRC.The level of ANXA4 autoantibody can reflect the prognosis of CRC patients receiving platinum-based chemotherapy with certain clinic application value.