| Hepatocellular carcinoma (HCC) is the fifth most common malignant tumor and the second leading cause of cancer-related deaths. A strong positive correlation has been demonstrated between HCC incidence rates and liver cirrhosis, and the risk of developing HCC for patients with HBV-induced cirrhosis is 1000 times that for individuals with non-HBV-induced cirrhosis. However, the interation of Glycan and proteins in the development of hepatitis, liver fibrosis/liver cirrhosis and HCC is still being investigated.Chapter 1 overviewed the information of glycomics and the future prospects of this study.Chapter 2 studied the alteration of membrane proteins in the development of HCC by lectin microarray. Lectin histochemistry was used to further validate the lectin binding profiles and assess the distribution of glycosidic residues in cells. In the fibrosis model,14 lectins (i.e., Jacalin, MAL-Ⅱ and AAL) were up-regulated while 3lectins (SJA, EEL and ConA) were down-regulated in the activated LX-2. In the HCC model,12 lectins (i.e., Jacalin, MAL-Ⅱ, LTL and LEL) showed increased signal while 6 lectins (i.e., WFA, PTL-Ⅰ, WGA and UEA-Ⅰ) showed decreased signal in HCC cell line HepG2; 11 lectins (i.e., Jacalin, MAL-Ⅱ, SJA and LTL) showed increased signal while 13 lectins (i.e., EC A, WFA, GSL-Ⅱ and PTL-I) showed decreased signal in HCC cell line SMMC-7721; Meanwhile, 10 lectins (i.e., ECA, GSL-Ⅱ, PTL-I and LEL) showed up-or down-regulated in the two HCC cell lines.8 lectins (i.e., ECA, GSL-Ⅱ, PTL-Ⅰ and LEL) showed increased signal while 2 lectins (Jacalin and VVA) showed decreased signal in HepG2 compared with SMMC-7721.The results of lectin histochemistry showed that the glycans were mainly localized to the cytoplasma membrane and perinuclear cytoplasm. In conclusion, the precision alteration of membrane protein glycosylation related to HCC may provide useful information to find new molecular mechanism of the development of HCC and anti-tumor therapeutic strategies.Chapter 3 studied the glycan-related genes expression in the the development of HCC, then the expression level of the altered glycan-related gene and proteins were conformed by Real-time PCR and Western blot, respectively. In the fibrosis model, expression level of 39 probe sets were higher, while 36 probe sets were lower in the activated LX-2, the expression profile detected the presence of gene transcripts in each sample for approximately 18.66% (75) of all probe sets contained in the microarray. However, the glycan-related gene microarray contained probe sets to monitor the expression of 190 glycan-related genes transcripts listed above, and the expression profile detected the presence of approximately 22.11% (42) of all glycan-related genes transcripts in the activated LX-2. In HCC model, expression level of 29 probe sets were higher, while 37 probe sets were lower in HepG2; expression level of 19 probe sets were higher, while 14 probe sets were lower in SMMC-7721. The expression profile detected the presence of gene transcripts in each sample for approximately 67.66% (272±93) of all probe sets contained in the microarray. However, the glycan-related gene microarray contained probe sets to monitor the expression of 190 glycan-related genes transcripts listed above, and the expression profile detected the presence of approximately 60.00% (114±2) of all gly can-related genes transcripts in HCC cell lines. Our aim of the study was to investigate potential associations between glycan-re la ted genes expression and glycan profiles to evaluate liver fibrosis in the development of HCC.Chapter 4 studied the alteration and localization of glycan-binding proteins (GBPs) in the development of HCC by carbohydrate microarray. Carbohydrates cy/histochemistry was used to validate the results of the carbohydrate microarrays as well as assess the distribution and localization of their binding proteins in HCC cell lines and liver tissues. In the fibrosis model,12 carbohydrates (i.e., Gal, GaNAc and Man-9Glycan) showed increased signal, while 7 carbohydrates (i.e., NeuAc, Lac and GlcNAc-O-Ser) showed decreased signal in the activated LX-2; In the HCC model,8 carbohydrate probes (i.e., SL, LNT, and GalNAc) showed increased signal while 5 carbohydrate probes (li.e., Man, Man-9-Glycan, and Xyl) showed decreased signal in HepG2 compared with L02 cell line. The results of carbohydrates cy/histochemistry showed that GBPs mainly distributed in the cytoplasma membrane and perinuclear region of cytoplasm. The immunocytochemistry was further used to verify some GBPs really exist in Golgi apparatus of the cells. The precision alteration and localization of GBPs referred to pathological changes in HCC may provide pivotal information to help understand the biological functions of glycans how to exert through their recognition by a wide variety of GBPs. This study could lead to the development of new anti-tumor strategies and find the GBPs-related potential biomarkers.Chapter 5 studied the GBPs-related potential biomarkers in the development in HCC. In this part, two GBPs (GalNAc-binding proteins, GNBPs; Xylose-binding proteins, XBPs) were selected and subsequently isolated, identified and annotated. As a result of GNBPs, 264 GNBPs from the activated LX-2 and 257 GNBPs from the quiescent LX-2 were identified and annotated. A total of 46 GNBPs were estimated to be significantly up-regulated and 40 GNBPs were estimated to be significantly down-regulated in the activated LX-2. The GNBPs (i.e., BTF3, COX17, and ATP5A1) responsible for the regulation of protein binding were up-regulated, and those (i.e. FAM114A1, ENO3, and TKT) responsible for the regulation of protein binding were down-regulated in the activated HSCs. The motifs of the isolated GNBPs showed that Proline residue had the maximum preference in consensus sequences. The results of XBPs,70 XBPs from the activated HSCs and 64 XBPs from the quiescent HSCs were isolated, identified and annotated. A total of 30 XBPs were up-regulated (all fold change>1.5, p<0.05) and 14 XBPs were down-regulated (all fold chang≤0.67, p≤0.05) in the activated LX-2. The XBPs were localized at the cytoplasm and cytoplasmic membrane in LX-2 and cirrhotic liver tissues by cy/histochemistry. The XBPs (i.e., PDIA6 and CFL2) responsible for the regulation of protein binding were up-regulated, and those (i.e., TUBB and MX1) responsible for the regulation of catalytic activity were up-regulated in the activated HSCs. Then,2 candidates (e.g., PDIA6 and APOA1) were selected for further verification in the sera of HBV-induced chronic hepatitis/cirrhosis using western blotting and serum microarrays. Assessments of PDIA6 and APOA1 as biomarker candidates show a higher discrimination (Area Under Curves, AUCs=0.8985,p＜0.0001) relative to APOA1 (AUCs=0.8738, p<0.000\) in sera of patients. In conclusion, the precision alteration of the XBPs referred to pathological changes in LX-2 during liver fibrosis/cirrhosis may provide pivotal information to discover the potential GBPs related bio markers for diagnosis of liver fibrosis/cirrhosis and development of new anti-tumor strategies.Chapter 6 studied the function of glycans in the population living with chronic diseases. In our recent study, we found that saliva proteins may protect older people from influenza, however, it is often noted that hospitalizations and deaths after an influenza infection mainly occur in the elderly population living with chronic diseases, such as diabetes and cancer. Our objective was to investigate the expression level of the terminal a2-3-and a2-6-linked sialic acids in human saliva from type 2 diabetes mellitus (T2DM), liver disease and gastric cancer (GC) patients and assess the binding activity of these linked sialic acids against influenza A viruses (IAV). We observed that the expression level of the terminal a2-3-linked sialic acids of elderly individuals with T2DM and liver disease were down-regulated significantly, and the terminal a2-6 linked sialic acids were up-regulated slightly or had no significant alteration. However, in the saliva of patients with GC, neither sialic acid was significantly altered. These findings may reveal that elderly individuals with chronic diseases, such as diabetes and liver disease, might be more susceptible to the avian influenza virus due to the decreased expression of terminal α2-3-linked sialic acids in their saliva. |
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