Quantitative Proteomics Study Of Tissue-derived Secretome Of Primary Hepatocellular Carcinoma(HCC)

Objective By using iTRAQ-label-based quantitative proteomic technique,the present study aims to explore the characteristics of bioinformatics of differential proteins among HCC tissues,adjacent tissues and distant carcinoma tissues.Besides,GO and IPA analyses were employed to have a preliminary understanding of the molecular basis of the occurrence and development of HCC.At last,serum concentration of differentially expressed secretory proteins(DEPs)were analysed to explore the relationship between DEPs and HCC.Methods 1.The HCC tissues,adjacent tissues and distant carcinoma tissues were cultured in serum-free medium in vitro and the secretory proteins were extracted.the secretory proteins of different time points were analysed by HE and Tunel analysis to determine the suitable culture time,and Western blot was employed to analyse the contamination of non-secretory protein.2.Using the iTRAQ combined with 2D LC-MS/MS technique,we identified and screened the DEPs,and analyzed the proteins in order to have a preliminary understanding of the characteristics of proteomics secreted by HCC tissues.3.GO analysis and IPA analysis were performed on 102 DEPs between HCC tissues and distant carcinoma tissues,between adjacent tissues and distant carcinoma tissues.4.The preoperative serum of HCC patients and matched healthy people were collected and analyzed by PRM analysis.At the same time,the serum of 82 HCC patients after hepatectomy were analysed by ELISA.The serum concentration of CA2 and KRT19 were analysed by ROC and survival analysis to illuminate the diagnosis and prognosis ability of CA2 and KRT19.Moreover,the relationship between the serum concentration of CA2 and KRT19 and the clinical and pathologic characterics of HCC was analyzed by Logistic regression analysis.Results 1.We find that the best time to extract secreted proteins is that when the tissue was cultured for 1 day.At that time,the WB result shows that there is basically no contamination of intracellular proteins.2.There are 2388 secreted proteins identified by proteomics analysis,and all of them meet the proteomic characteristics of secreted proteins.Compared carcinoma tissues with distant carcinoma tissues,there are 36 DEPs.Among them,14 proteins expression were up-regulated and 22 were down-regulated.Compared adjacent tissues with distant carcinoma tissues,there were 90 DEPs.Among them,27 were up-regulated and 63 were down-regulated.The two groups have 24 common DEPs.Among them 11 were up-regulated and 13 were down-regulated.3.The bioinformational analysis of the differentially expressed proteins shows that the proteins of HCC tissues,adjacent tissues and distant carcinoma tissues are paticipated in different BP and MF.The DEPs of C/F group are associated with metabolic progress,organ development and tumor microenvironment.The DEPs of P/F group are associated with metabolic progress and coenzyme metabolic progress.IPA analysis shows that the DEPs are associated with ERK,AKT/PI3 K and NF-?B signal pathways,Besides,both CA2 and KRT19 are involved in three network pathways.4.Using PRM analysis to analyze the serum concentration of CA2 and KRT19 in HCC patients and healthy people,we find that serum concentration of CA2 and KRT19 of HCC patients are significantly higher than those in healthy people.The ELISA technique was used to examine the serum concentration of CA2 in 82 HCC patients underwent radical resection.ROC analysis shows that the AUC of CA2 and KRT19 are 0.715 and 0.728,combination of CA2 and KRT19 has a reliable diagnostic performance for HCC(AUC=0.817,sensitivity=67.3% and specificity=87%).The serum concentration of CA2 is significantly associated with the number of tumor(P=0.026)and BCLC staging(P=0.042).CA2 is an independent risk factor for recurrence/metastasis after HCC radical resection(P<0.05).The recurrence-free survival of HCC patients with low-expressed of CA2(CA2?41.8ng/m L)is significantly higher than those with high-expressed of CA2(P<0.05).Conclusions 1.We establish the culture system of HCC tissue in vitro successfully.It is feasible to culture hepatoma tissue in vitro and extract the secreted proteins.2.There are differences in the secretory proteins among the three tissues,HCC tissues,adjacent tissues and distant carcinoma tissues according to iTRAQ-based proteomics analysis.These differential proteins could be used as potential alternative markers for the early diagnosis of HCC.3.Metabolic disorders are closely related to the occurrence of HCC,the disorders of energy metabolism and ethanol metabolism playe an important role in the occurrence and development of HCC.CA2 and KRT19 may promote the development of HCC through the network pathway of ERK,AKT/PI3 K,NF-?B protein-protein interaction.4.CA2 and KRT19 are biological targets,both of them can be used for the diagnosis of HCC.CA2 can also be used for the prediction of recurrence and metastasis of HCC patients and the evaluation of tumor-free survival.