Analysis Of Glycosylation On Serum Glycoproteins And Application On The Diagnosis Of Primary Liver Cancer

Primary liver cancer(PLC)is one of the major types of malignancies greatly threatening human health.In China,PLC is the fourth most common tumor with the third highest mortality rate.As most of the patients were diagnosed at advanced stage,both of postoperative recurrence and metastasis rates were so high that the long-term life quality is poor and the five-year survival rate is dismal.Early diagnosis and early intervention are crucial for PLC patients.Serum alpha-fetoprotein(AFP)is the most commonly used tumor biomarker of PLC.However,the sensitivity of AFP is only about 60-70%,and increased AFP levels can also be detected in some benign liver diseases.Therefore,the diagnostic efficacy of AFP cannot meet clinical needs.To find more powerful biomarkers or multiparameter diagnostic model is an important direction for the study of PLC.In addition,noninvasive serum markers which can be used to evaluate or predict the key pathological factors affecting the prognosis of PLC are in urgent need for individualized diagnosis and treatment decision making.Previous studies of tumor biomarkers mainly focused on proteins and nucleic acids,attaching less significance to sugar chains or glycans,which is an important type of biological polymer.The covalent attachment of glycans to specific amino acid residues of the polypeptide backbone is the so-called protein glycosylation,which includes two main types,namely,N-glycosylation and O-glycosylation.About 70% of proteins in human body are glycosylated.Glycosylation is the most common and most complicated post-translational modification,and it plays key regulatory roles in the structures and functions of proteins.Glycosylation is involved in a series of biological processes such as cell adhesion,cell-cell interactions,signaling,and immune regulation etc.A lot of studies have demonstrated that glycans are almost directly involved in the pathophysiology of all major diseases in humans,including tumors.Glycoscience provides a new perspective for disease-related researches.Glycomics,glycoproteomics and glycobiology have been a hot topic of broadly concern.In fact,most commonly used tumor biomarkers are glycoproteins,including AFP,carcinoembryonic antigen(CEA),prostate specific antigen(PSA)etc.Therefore,it is of great value to study cancer-related glycosylation changes for cancer diagnosis,monitoring,prognosis and even targeted therapy.However,the complexity and diversity of glycans is a great challenge for structural and functional studies.The development of glycoscience lags far behind genomics and proteomics.In this study,the prevailing techniques including mass spectrometry and capillary electrophoresis were adopted to analyze N-glycans released from specific proteins or from total serum proteins to explore the clinical value of N-glycans in PLC.The analytic method for characterization of intact and micro-heterogeneous N-glcopeptides was also studied.In addition,the underlying regulatory mechanisms of aberrant glycosylation was explored through transcriptome sequencing.Part Ⅰ: Analysis of N-glycans derived from serum IgG and its diagnostic value in HCCIn this part,we used the matrix assisted laser desorption/ionization mass spectrometry(MALDI-MS)to identify and compare the N-glycans released from serum IgG of hepatocellular carcinoma(HCC)and liver cirrhosis(LC)patients.The results showed that the main glycoforms of IgG changed during the development of HCC.Generally speaking,compared with the LC patients,galactosylation levels of the N-glycans on IgG of HCC patients were decreased and were related to etiology.In HCV related HCC samples,some N-glycan structures were associated with clinicopathological characteristics such as tumor size and TNM staging.In addition,the N-glycans derived from serum IgG were of diagnostic value for HCC,especially for AFP negative HCC.Part Ⅱ: The correlation between serum N-glycan profiling and clinicopathological characteristics of HBV related HCC patientsIn this part,fast and high-throughput DNA sequencer-assisted flurophore-assisted carbohydrate electrophoresis(DSA-FACE)technology was used to analyze serum Nglycome of HBV related HCC patients,and to explore the relationship between N-glycans and clinicopathological characteristics.The results showed that relative abundance of serum N-glycans were related to some important clinicopathological factors such as microvascular invasion(MVI),tumor size,portal vein tumor thrombus(PVTT),cirrhosis and differentiation.In addition,the predictive efficacy of N-glycans and N-glycan based diagnostic model for MVI were not enough but also not lower than that of AFP.Analysis of follow-up data revealed that Peak12(NA4Fb,the branching alpha-1,3-fucosylated tetraantennary glycan)was an independent risk factor for postoperative recurrence,and Peak9’(NA3F,the core-alpha-1,6-fucosylated triantennary glycan)and Peak12 were independent risk factors for cancer-induced death.Part Ⅲ: LC-MS based site-specific N-glycopeptides analysisIn this part,alpha-1 antitrypsin(A1AT)and fibronectin were selected as the target protein.Liquid chromatography-mass spectrometry(LC-MS)were used to establish the method for site-specific N-glycopeptides analysis.The micro-heterogeneity of glycosylation was characterized on A1 AT and Fibronectin.At the same time,serum A1 AT was purified using immunoprecipitation from the patients with HCC and LC.Three differentially expressed N-glycopeptides were found between HCC and LC.Analysis of glycosylation micro-heterogeneity provided a more precise way for revealing glycosylation aberration during tumorigenesis.Part Ⅳ: Knockout of alpha-1,6 fucosyltransferase(FUT8)in SMMC-7721 cell line and transcriptome sequencingIn this part,we knocked out the gene of alpha-1,6 fucosyltransferase(FUT8)responsible for core fucosylated structures from SMMC-7721 cell line.Transcriptome sequencing was performed on Illumina Hiseq platform,and the RNA sequencing data were verified by real-time quantitative PCR(RT-PCR).Additionally,211 differentially expressed genes were identified and functionally annotated,and the major signaling pathways(PI3K/AKT et al.)were also enriched,providing clues for molecular regulation studies of abnormal glycosylation in HCC.To sum up,in the present study,a variety of methods were adopted to analyze the Nglycans of target protein,N-glycome of serum and intact N-glycopeptides of specific proteins in order to explore the aberrantly glycosylated structures associated with HCC,and pave the way for the clinical application of glycoscience in cancer.Additionally,the regulatory network of aberrant glycosylation was preliminary explored by transcriptome sequencing,and the related mechanisms need to be further investigated.