| Primary liver cancer(PLC)is one of the malignant tumors with the higher mortality rate,high aggressive and bad prognosis in our country.For PLC diagnostic rates,especially early PLC,is very low.The early diagnosis of PLC is very important.?-fetoprotein(AFP)is the most widely recognized biomarker by far.However,it is reported that the sensitivity of AFP is just 30.9%-75.0%with a specificity of 63.0%-99.5%.So more novel and more sensitive biomarkers are needed to be found.With large-scale analysis of whole protein expression,proteomics can study dynamic expression and functional changes of proteins related biological processes.It's a high-throughput method to find differential diagnostic biomarkers and study pathogenesis.It can find biomarkers that traditional means can not be obtained,so it became the main method of looking for new tumor markers.Glycomics is an emerging subject following the genomics and proteomics.Changes in serum protein glycosylation will affect its structure and function.Meanwhile,most of the markers belong to glycoproteins.So glycomics is a promising approach to screen novel biomarkers for the diagnosis of early PLC.In this study,serum samples from normal control,hepatitis B,liver cirrhosis and PLC groups were collected to screen differential proteins by proteomics.Later,part of these screened proteins was validated and further study.Then lectin microarray was used to establish the serum glycoproteome profiles of the four experimental groups.PART 1SCREENING OF SERUM PROTEIN MARKERS FOR VARIOUS STAGES OF PRIMARY LIVER CANCER GENERATING PROCESSESIn this part,the goal is to screen out the differential proteins with potential diagnostic value during various stages of hepatocellular carcinoma generating processes.iTRAQ and LC-MS/MS was used to analyze serum proteome alterations dynamically and quantitatively.First,serum samples from normal control(Nc),hepatitis B(HepB),liver cirrhosis(LC)and PLC were collected in serum specimen bank which was established by collecting serum samples from high incidence area of PLC in Guangxi.14 high abundance proteins were removed from serum samples and then concentrated.After all samples subjected to iTRAQ labeling followed by LC-MS/MS analysis,a total of 316 proteins were identified.These proteins were filtered with manually selected filter exclusion parameters:P<0.05,EF<2,proteins were considered up regulated when ratios>1.2 and down regulated when ratios<0.8.Thus 93 proteins were screened out as differential expressed proteins.There were 43,70,51 proteins showed differences in expression levels in HepB,LC,PLC with the comparison of Nc respectively.For these identified differential proteins,GO analysis was used to analyze the protein expression profiles data.The subcellular distributions were enriched in extracellular region(53%).Most differential proteins were involved in biological regulation(33%),metabolic process(25%)and immune(14%),protein binding(37%)and enzymic activity(35%)on molecular function.Western blot was used to validate CD 14,QSOX1,GELS and ELISA was applied to detect CD14,the results indicated the credibility of iTRAQ results.CD 14 was chosen for further study.Receiver operating characteristic(ROC)curve was estimated with a maximal sensitivity 94.7%and specificity 50%with cut-off 3.16ng/ml of CD 14 for the detection of PLC while sensitivity 31.6%and specificity 94.4%with cut-off 191.4ng/ml of AFP.CD 14 was available to distinguish LC and PLC(AUC=0.760,P<0.05).When two indicators combined,the diagnostic efficiency was increased distinctively(AUC=0.889,P<0.05)with sensitivity 84.2%and specificity 83.3%.The present results demonstrated that CD 14 was available to distinguish the LC and PLC,the diagnostic efficiency was increased when CD 14 was combined with AFP.So CD 14 was proved as a potential biomarker for the detection of early PLC.PART 2COMPARATIVE STUDY ON SERUM GLYCOPROTEOME PROFLING DURING THE VARIOUS STAGES OF PRIMARY LIVER CANCER GENERATING PROCESSES BY LECTIN AFFINITY MICROARRAYIn this part,we carried out a comparative research on serum glycoproteome profling during the various stages of hepatocellular carcinoma generating processes by lectin affinity microarray.A lectin array which has been validated was established in our laboratory.Such technology(contains 50 lectins)was used to identify specific serum glycoproteome profiles during the different stages of hepatocellular carcinoma generating progresses for three times.It found that the enhanced affinity on AAL,ACL,ConA,LCA,MPL,NML,PHA-E,PHA-L,PSA,RCA-I,STL,VAL,WGA,SNA(P<0.05)was assosiated with the carcinogenesis of PLC.The results implied the changes in specific glycan structures,such as ?Fuc,GlcNAc,GalNAc,mannose,bisecting GlcNAc,terminal ?1-4 Gal and so on.But it is no significant differences between PLC and HepB of SNA.The most likely reason for it might be the changes in the sialyltransferase activity during acute and chronic stress.Then lectin labeled ConA?LCA and PHA-E were further confirmed by lectin blot.The results were consistent to the lectin array,which indicated the credibility of lectin array results.This study indicated that the altered glycan structures may have poteintial diagnostic value,which provided important information for us to find a novel biomarker of early PLC.CONCLUSIONS1?iTRAQ labeling followed by LC-MS/MS analysis was used to study the dynamic of proteome during the various stages of hepatocellular carcinoma generating processes,a total of 316 distinct serum proteins were identified and quantified.There were 43,70,51 proteins showed differences in expression levels in HepB,LC,PLC with the comparison of Nc respectively.2?CD14 was available to distinguish LC and PLC(AUC=0.760,P<0.05).The efficiency of early PLC diagnosis was increased when CD14 was combined with AFP(AUC= 0.889,P<0.05).CD 14 was proved as a potential biomarker for the detection of early PLC.3?14 enhanced affinity of lectins were detected by lectin microarray,which indicated that glycoproteins with these changed glycan structres may be the potential biomarkers for the detection of early PLC.NOVELTY OF PROJECT1?Serum samples were collected from a serum specimen bank established through sreening and regular follow-up high risk queue from high incidence area of PLC in Guangxi.Therefore the dynamic observation of serum related indexes changes for the high-risk population of liver cancer can be obtained.2?iTRAQ labeling followed by LC-MS/MS analysis was used to study the dynamic of proteome during the various stages of hepatocellular carcinoma generating processes.CD14 was proved as a potential biomarker for the detection of early PLC.3?Serum glycoproteome profiling was established during the various stages of hepatocellular carcinoma generating processes by lectin affinity microarray.THE POTRNTIAL APPLICATION OF THIS WORKCD 14 was available to distinguish LC and PLC,so CD 14 was proved as a potential biomarker for the detection of early PLC. |
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