Comparison Of Serum Glycoproteomics Between Human Non-Metastatic And Metastatic Hepatocellular Carcinoma

Objective : The recent developments of glycoproteomics and glycomics technologies allow us to analyze the serum glycoproteins from the patients bearing hepatocellular carcinoma (HCC) with different degree of malignancy.By the dynamic changes of protein and sugar levels we explored the molecular markers and discuss the characteristics of metastatic HCC.Background:Proteomics technologies analyze the structure and function of proteins of the organism from the overall perspective, containing the dynamic changes of components, expression levels, modification status and the interactions. Protein expression, protein post-translational modification state and subcellular localization are important aspects to the regulations of biological functions. Hence, the quantitative comparison of protein expression and their modifications are important for studying the mechanism of diseases during their progression and development.The"proteomic pattern diagnostics"approach to the discovery of new biomarkers is based on two premises: (a) the low abundance serum proteome contains an enormous wealth of biomarker information, which has not yet been explored, and (b) a multiple biomarkers diagnostic model may definitely have high sensitivity and specificity with confidence than that of a single biomarker alone across large heterogeneous patient populations, and for complex multistage diseases such as cancer^[1]. In recent years, Glycomics and proteomics in the cancer research field are rising. A large number of studies have shown that glycosylation of proteins and their corresponding functional role will change in the development and invasion process of tumor ^[2-5]. Recently, according to reports, the lentil lectin LCA-bound alpha-fetoprotein (AFP-L3) test has gained FDA approval as a risk marker for primary liver cancer. Compared to alpha-fetoprotein (AFP), AFP-L3 have been approved the sensitivities and specificities ranging from 30.9% to 75% and 63% to 99.5%, respectively in the detection of HCC^[6-8].Therefore, through the application of proteomics combined with glycomics research strategies allow us to understand the mechanisms of cancer and its metastasis more comprehensively.Primary liver cancer is the fifth most frequent neoplasm and it is the third most common cause of cancer death, with more than 500,000 new cases diagnosed yearly^[9],which is common in China and Asia. At present, some commonly used biology characteristic indicators of tumor, such as alpha-fetoprotein (AFP), tumor size and vascular invasion are not accurate enough to diagnose, predict and distinguish the malignant of liver cancer. The proteomics researches associated with the treatment and occurrence of liver cancer are arising^[10-12]. However, there is a few proteomics research on HCC metastasis, especially in the area of glycomics.Metastasis is the primary factor of survival in cancer patients. Someone has considered that overcome the problems of "tumor metastasis" will be the most urgent need in life sciences during this century.Combining the proteomic with glycomic techonolgy,we explore the dynamic changes in protein expression levels and glycosylation levels of serum proteins from patients with different degrees of malignancy of HCC. We attend to screen the new serum molecular markers of metastatic HCC.Part oneComparison of glycan profiling of serum glycoprotein between non-metastatic and metastatic Hepatocellular carcinoma by lectin affinity technologyMethods: We collected sera from 36 hepatocellular carcinoma patients which contain 18 TNMⅠ-Ⅱhepatocellular carcinoma patients (non-metastatic HCC group) and 18 TNMⅢ-Ⅳhepatocellular carcinoma patients (metastatic HCC group), according to 2002 UICC TNM staging system. Every 6 hepatocellular carcinoma patients's sera are pooled. Serum high abundance proteins are depleted by using column ProteoExtractTM Albumin / IgG Removal kit and protein concentration is determined with 2-D Quant Kit. The high abundance proteins depleted serum proteins were labeled with fluorescence dye Cy5. The tumor specific lectin microarray containing thirteen kinds of lectins were employed in this assay. Lectin affinitied glycan profiles of serum glycoprotein between non-metastatic HCC and metastatic HCC were compared and differential lectin affinitied glycans were further verified by multi-lectin blotting.Results: Both lectin microarray and lectin boltting assay showed enhanced affinity on WGA, SNA, PHA-E, DSA in metastatic HCC serum proteins(P<0.05).Conclusion: It showed that the specifc glycan profiles of metastatic HCC serum glycoprotein involved increased Sia, GalNAc ,Multi-antennary structure, polyLacNAc and LacNAc(NA2 ,NA3, NA4). The results also indicated that the alternated sugar chains might be associated with tumor invasion and metastasis .Part TwoThe quantitative proteome anaysis of WGA affinitied serum glycoproteins between non-metastatic and metastatic Hepatocellular carcinomaMethods: We collected sera from 36 hepatocellular carcinoma patients , 18 patients belong to TNMⅠ-Ⅱ(non-metastatic HCC group) and 18 belong to TNMⅢ-Ⅳ(metastatic HCC group), according to 2002 UICC TNM staging system. Every 6 hepatocellular carcinoma patients's sera are pooled. The control group 27 cases of healthy people are pooled as a mixture serum sample. Lectins are usually used for the enrichment of glycoproteins.We selected the lectin of wheat germ agglutinin (WGA) to enrich serum glycoproteins . WGA has the binding specificity to the terminal glycan epitopes of (GlcNAc) n andα2, 3/6/8 type of sialic acid on glycoprotein.With WGA wheat germ agglutinin chromatography, the WGA binding glycoproteins are labled with mass-balanced isobaric tags(iTRAQ) and analyzed by liquid chromatography-tandem mass spectrometry(LC-MS/MS). The differential protein of SERPINA1 were further verified by Western blot method on protein levels .Results: With the technology of iTRAQ labeling followed by liquid chromatography tandem mass spectrometry (liquid chromatography of a tandem Mass spectrometry, LC-MS / MS) analysis , 15 differential serum WGA affinited glycoproteins were finally screened out. Among them, 9 proteins were up-regulated (>1.5-folds) and 6 were down-regulated(>0.5-folds) in HCC patients with metastasis.Protein SERPINA1 which has been verified at the protein expression level, indicating that the protein levels in metastatic HCC group is increased.Conclusion: Our study has obtained a set of HCC metastasis associated glycoproteins which may serve as novel prognostic candidates and potential therapeutic targets for HCC metastasis. The potential value of these specific glycan aberrations as HCC metastasis biomarkers is worth for further studying.Novelty1. Previous studies on liver cancer were mainly used cell lines or animal models with different metastatic potential. Our study is the first time to compare the serum glycoprotein profiling from the HCC patients with and without metastasis.2. The lectin microarray technology and multiple lectin boltting assay revealed enhanced serum glycoprotein affinity of WGA, SNA, PHA-E, DSA in metastatic HCC patients compared to non-metastatic HCC(P<0.05). 3. Based on iTRAQ labeling followed by liquid chromatography tandem mass spectrometric analysis , 15 differentially WGA binding proteins in metastatic cases has been screened out with comparision of non metastatic HCC patients. Among them, 9 proteins were up-regulated(>1.5-folds) and 6 were down-regulated (>0.5-folds). The relation of SERPINA1 with metastasis of HCC was first reported.Potential ApplicationLectin based technology is relatively new and feasible research strategy for glycoproteomics and glycomics.The study screened a set of HCC metastasis associated glycoproteins which may serve as new prognostic candidates or potential therapeutic targets for HCC metastasis . The potential value of these specific glycan aberrations as HCC metastasis biomarkers is worth further studying.