| Hepatocellular carcinoma (HCC) is one of the most common cancers worldwideand has a high mortality rate due to the difficulty of early diagnosis and limitedtherapeutic methods. Therefore, it becomes critical to reveal the underlyingmolecular mechanisms of HCC development and to look for early diagnosticbiomarkers and treatment targets for HCC.The glycans of glycoproteins play pivotal roles in cell recognition, signaltransduction, immune surveillance and maintenance of ordered life activities ofmutilcellular organisms. Glycan alternations are closely associated with theinitiation and progression of many diseases including cancer. Research onstructures and functions of tumor-specific glycans will not only reveal thebiofunctions of glycans, but also enable the identification of new cancer biomarkersand drug targets. DSA-FACE (DNA sequencer-assisted fluorophore-assistedcarbohydrate electrophoresis) is an emerging glycobiological technique in thiscentury and has been widely used in the research of serum biomarkers for cancer.However, the application of this technique in non-serum specimens has rarely beenreported.This thesis studied the structure of HCC-specific N-glycans and associatedmolecular mechanisms using DSA-FACE technique. First of all, DSA-FACEtechnique used to detect N-glycans in serum was optimized and the detectivequantum efficiency has been remarkably promoted. Based on this, a DSA-FACEtechnique that can detect N-glycans in the in-vitro cultured cell lines and humanclinical tissues was developed. Compared to the conventional DSA-FACE, thisnewly developed technique has much higher stability, sensitivity and repeatability,which has been achieved by optimizing membrane protein extraction methods andintroducing GC resin purification procedure. This technique expands theapplication field of DSA-FACE and eliminates the limitation of conventionalDSA-FACE technique that only can be used for the glycan detection in serumsamples.Using the optimized serum DSA-FACE technique,190serum samples of HCCpatients were detected along with179serum samples of healthy humans and110serum samples of HBV-infected patients as controls. The results indicated thatLog[(NGA2F+NGA2FB)/NA3] which composed of3abnormal N-glycans, NGA2F,NGA2FB and NA3, in HCC patients are potential early diagnostic serumbiomarkers for HCC. The diagnostic efficiency of this biomarker is as high as85%with71.1%sensitivity and86.0%specificity. The diagnostic efficiency of this biomarker is independent of sex, age, tumor stage and differentiation degree ofHCC patients. Furthermore, the serum samples from23out of40HCC patientswhose AFP values are within normal range (AFP <20ng/ml) showed distinctlyhigher Log[(NGA2F+NGA2FB)/NA3] value above the threshod (0.1045), whichindicated that Log[(NGA2F+NGA2FB)/NA3] can effectively reduce the falsenegative rate of AFP detection. Thus, a HCC-specific glycans (NGA2F, NGA2FBand NA3)-AFP combined diagnosis model was further established using Logisticregression. The diagnostic efficiency of the combined diagnosis model for HCCreached94.8%as shown in ROC curve. This result supports the idea that the serumN-glycan biomarker-based combined diagnosis model can serve as a HCC-specificscreening biomarker in the north of P. R. China.Then, the cell surface N-glycan profiles of88pairs of HCC clinical tissues wereexamined by the newly established DSA-FACE technique which was suit for celllines and clinical tissues. The results indicated that the N-glycan peak L10(corresponding to NA3FB) was significantly increased (p<0.0001) on the cellsurface of HCC tissues compared to the paired adjacent normal tissues, while peakL6was significantly decreased (p<0.0001) on the cell surface of HCC tissues. Sametrend of alternations on both N-glycans were observed in all sex, age and celldifferentiation degree groups. In fact, these alterations have been found to presentat early stage of HCC. In addition, by comparison with the N-glycan profiles of100pairs breast cancer tissues we found that NA3FB was significant increased on thecell surface in a tissue and sex independent manner, while NA2and M8showeddistinct tissue specificity. The further comparative analysis of the clinical tissuesand corresponding serum samples in61HCC patients showed that NA3FB was notonly increased on the cell surface of HCC cells (p<0.0001), but also increased inthe corresponding serum samples (p<0.0001). This indicated that the abnormalincreased resident and excreted NA3FB glycoprotein was likely related to thetumorogenesis mechanisms of HCC.In order to investigate the molecular mechanisms of the significant enhancedNA3FB, both resident and excreted glycoproteins, in HCC patients, the expressionlevel of5glycosyltransferases (Fut8, GalT-1, GnT-V, GnT-IVa, GnT-IVb) whichcan produce NA3FB in HCC were analyzed. The results indicated that onlyGnT-Iva on both mRNA and protein levels were significantly increased in HCCtissues compared with paired adjacent tissues. The cell surface NA3FB in HepG2cells was detected48hours later after mgat4a (the gene of GnT-IVa) knockdown byusing RNAi technique. NA3FB was significantly decreased in response to mgat4aknockdown, suggesting that the decrease of NA3FB was related to the gene silence of GnT-IVa. Therefore, the enhanced GnT-IVa in HCC was closely related to theincreased levels of NA3FB both on HCC cell surface and in serum.To sum up, this study successfully established a N-glycan DSA-FACE techniquewhich was suit for the analysis of different clinical tissue specimens. The obtainedN-glycan serum biomarker can be used for the early diagnosis of HCC in the northarea of China. HCC-cell-surface-specific biomarker NA3FB was closely associatedwith the increased GnT-Iva in HCC. Therefore, the preliminary study on this alertedglycosylation mechanisms in HCC provides a new path for further research on therelationship of glycosyltransferases and glycan structure with the carcinogenesisand malignant progression of HCC. It also advanced the study on the earlydiagnosis and targeting therapy of HCC. |
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