| Primary liver cancer ranks the sixth most common cancer but the third frequent cause of cancer death in the world. In China, it accounts for the second major cause of cancer death. Among primary liver caners, Hepatocellular carcinoma (HCC) represents the major histological subtype, accounting for 70%-85% of the total liver cancer burden worldwide. Hepatic resection remains the main strategy to obtain long-term survival for HCC patients currently. However, even after radical resection of small hepatocellular carcinoma, there are still more than 60% of patients undergoing metastasis or recurrence in 5 years. Obviously, recurrence and metastasis after radical resection has become one of the major obstacles for long-term survival of HCC patients. Therefore, in order to increase the survival rate, it is critical to reveal the molecular mechanism of metastasis and recurrence, and explore predicting biomarkers for metastasis and recurrence of HCC patients after radical resection. Much effort had been made in this field by the means of both genomic and proteomic ways, but the results are still unsatisfied.Glycosylation is an important post-transcriptional modification, which is the key procedure for maturing and obtaining biological function of protein. Most secretory proteins and nearly all immunity-related proteins are glycoproteins, whose changes are tightly associated with tumorigenesis and invasion of malignant tumors. Moreover, a lot of biomarkers for malignancies are glycoproteins, such as AFP, CEA, CA125 and PSA, etc.. Obviously, glycoproteomics plays a pivotal role in the research field of malignancies. Recent studies have shown that glycoproteins could be served as prognostic biomarkers and therapeutic targets of HCC.This study aims to develop an integrated strategy for high-throughput quantitative comparison of N-glycoproteins. This strategy could generate quantitation ratio of the glycopeptides and glycoproteins accurately and reliably, and reflected the changes in glycoprotein of various glycan types in complex biological samples precisely. We also utilized this strategy in analyzing the potential metastasis and recurrence-related glycoproteins in serum samples from early HCC patients (BCLC stage 0-A), attempting to analyze metastasis and recurrence-related changes in glycoproteins and explore their potential applications as glycan-relevant biomarkers.PART IDeveloping a new quantitative method of N-glycoproteome using lectin affinity combined with enzyme-catalyzed 18O3-labelingBackground Complexity of protein glycosylation makes it difficult to characterize glycosylation patterns on a proteomic scale. In this study, we developed an integrated strategy in an attempt to quantitatively analyze altered N-glycoproteins and N-glycosylation from complex biological samples in a high-throughput way.Methods This strategy includes using lectin chromatography to enrich and separate the glycopeptides/glycoproteins, tandem labeling 18O and 16O that generated a mass shift of 6 Da between paired labeled glycopeptides, and analyzing glycopeptides with LC-MS and self-built automatic quantitative method based on Mascot Distiller. The accuracy and repeatability of this quantitative method was verified by standard glycoprotein, and after that, the integrated strategy was applied in serum samples of HCC patients and healthy individuals..Result The automatic quantitative method could maintain linearity within the range of 1:10-10:1 with the correlation coefficient r2>0.99. Peptide concentration ratios calculated by self-built quantitative method were similar to the theoretical values and the ratios from manual calculation. For the glycopeptide, the SD range was identified as 0.023-0.186. The results showed that 43 glycopeptides and 35 glycoproteins were significantly different between HCC patients and healthy individuals; one of them was confirmed by western blot assay.Conclusions The integrated strategy is accurate, repeatable, and highly effective due to the introduction of the automatic quantitative method. This integrated strategy provides a useful tool for screening N-glycosylation and N-glycoproteins as diagnostic and prognostic glyco-biomarkers.PART IIQuantitative serum N-glycoproteome analysis on prognostic role of differential N-glycoprotein expression in early hepatocellular carcinomaBackground Glycoproteins played an important role in HCC invasions and metastasis. We analyzed recurrence-related changes in glycoproteins of various glycan types and explored their potential applications as glycan-relevant biomarker by the new self-built quantitative strategy of N-glycoproteome in serum samples from early HCC patients (BCLC stage 0-A).Methods We preliminarily identified the changes of glycan types and corresponding lectins related to HCC recurrence, by using lectin microarray and glycoprotein gel staining method in paired tumor and peritumor tissue samples (40 cases) from early HCC patients (BCLC stage 0-A). Combining with our new lectin affinity and enzyme-catalyzed 18O3-labeling N-glycoproteomics strategy, we screened recurrence-related changes in serum glycoproteins from early HCC patients (40 cases). We verified 5 changed glycoproteins by western blot in another serum samples (60 cases), and analyzed theirs predictive value for prognosis, evaluated the relationship between these proteins and overall survival or recurrence in early HCC patients. Finally, these findings were validated in HCC cell lines.Result We found some recurrence-related lectins in tumor and peritumor tissues from early-stage HCC patients by lectin microarray and glycoprotein gel staining method. In HILIC, ConA, LCH, and WGA enriched N-glycoprotein subgroup, we identified 44 glycoproteins changed in both total glycoprotein subgroup and every lectin subgroup. Among these,16 glycoproteins abundance changed in multi-subgroup. We validated MASP1, QSOX1, Ceru, LG3BP, and Thrombin protein by western blot. Expression level of LCH enriched QSOX1 (al-6 fucosylated QSOX1) in serum of the recurrence group was significantly lower than those without recurrence. The expression level of al-6 fucosylated QSOX1 in serum was independent factors affecting recurrence by univariate (HR= 0.452, 95%CI=0.211-0.972; p=0.042) and multivariate (HR= 0.458,95%CI=0.213-0.988; p=0.047) Cox regression analysis using the median as cut-off point, but not related to overall survival. Similarly, the expression of QSOX1 in HCC cell lines reduced with the invasive ability of HCC cell lines increased.Conclusions The expression level of α1-6 fucosylated QSOX1 glycoprotein in serum is related to postoperative recurrence in early-stage HCC patients,α1-6 fucosylated QSOX1 is a potential glyco-biomarker to predict recurrence after radical resection of early-stage HCC patients. The expression of QSOX1 protein reduced with the invasive ability of HCC cell lines increased, and that meant it was a protective factor in HCC recurrence.Conclusions1. Our new quantitative method of N-glycoproteome using lectin affinity combined with enzyme-catalyzed 18O3-labeling is accurate and repeatable, and it can combine the changes of glycan types and specific glycoprotein abundance in complicated samples. This method is conducive to the discovery of biomarker and therapy target of disease.2. Our self-built quantitation method based on Mascot Distiller software, can provide a fast, accurate, reliable method to analyze the quantitative 18o3-labeling data. It can replace the time-consuming manual calculation used in our and other 18O3 labeling method to analyze the complex biological samples.3. The expression level of al-6 fucosylated QSOX1 glycoprotein in serum is related to postoperative recurrence of early HCC patients, and it is a potential glyco-biomarker to predict recurrence after radical resection in early HCC patients.4. The expressions of QSOX1 reduced with the invasive ability of HCC cell lines increased. QSOX1 protein is a protective factor in HCC recurrence.Novelties1. We developed a new quantitative method of N-glycoproteome using lectin affinity combined with enzyme-catalyzed 18O3-labeling, and finally confirmed that it is accurate and repeatable. It could be used for the study of complex clinical samples.2. We developed a new self-built quantitation method based on Mascot Distiller software. It could replace manual calculation, provided a fast, accurate, reliable quantitative data for our and other 18O3 labeling method.3. We firstly demonstrated that serum level of al-6 fucosylated QSOX1 glycoprotein was significantly lower in the recurrence group than without recurrence in early-stage HCC patients, and it was a potential glyco-biomarker to predict HCC recurrence.4. We firstly confirmed that the expression of QSOX1 protein reduced with the invasive ability of HCC cell lines increased, and it was a protective factor in HCC recurrence.Potential application of this study1. The serum level of al-6 fucosylated QSOX1 glycoprotein can predict the prognosis of HCC patients.2. Unraveling the pathological mechanism of QSOX1 in recurrence and metastasis of early-stage HCC patients may help to develop new therapeutic strategies for the prevention and treatment of recurrence and metastasis in these patients.3. The new quantitative method of N-glycoproteome using lectin affinity combined with enzyme-catalyzed 18O3-labeling can be used in glycoproteomics research on the discovery of biomarker and therapy target in other disease. |
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