| Recently, the International Agency for Research on Cancer (IARC) indicated that Hepatocellular carcinoma (HCC) is the fifth most common cancer in human worldwide, in2008, there are estimated749000new cases and695000deaths, of which402000cases and372000deaths occur in China (http://globocan.iarc.fr/) The China Statistical Year Book2010of MOH showed that malignant carcinoma has been the most common cause of disease death and HCC ranks the second place of all cancer deaths in China (http://www.moh.gov.cn). Presently, the diagnosis of HCC is usually achieved at a late stage, which results in the high mortality of HCC and an average survival time of less than12months for HCC patients after clinical diagnosis. Although serum alpha-fetoprotein (AFP) is commonly used marker for diagnosis of HCC, it usually causes false-positivity and false-negativity due to low sensitivity and specificity. About20%of patients with advanced HCC still showed negative for AFP measurement until they died. In China, more than90%of primary liver cancer is HCC.Protein glycosylation is one of the most important post-translational modifications, and glycoprotein glycans play a very important role in many physiological and pathological conditions. Disease-related glycomic studies show that glycan structures of glycoprotein have changed in the occurrence and development of many tumors, including HCC, and then they further lead to dysfunction of the glycoprotein and malignant transformation of cells. Therefore, the analysis of glycan structural change associated with the development of HCC and search for characteristic glycan markers of HCC will contribute to the early detection of liver cancer. Current studies have demonstrated that the deregulation cell cycle is closely associated with the development of tumors, and the glycosylation change can affect cell cycle regulation and cell proliferation and promote the development of tumors. Therefore, the present study focuses on the glycan structure changes in cell cycle progression of human hepatoma cell. Through the following three-part study, this study aimed to screen the characteristic glycopatterns in cell cycle progression of human hepatoma cell line.Part1:The establishment of the synchronization methods for human hepatoma cell line (HepG2) and normal human liver cell line (L02)Based on the existing cell synchronization methods and through the exploration and optimization of experimental conditions, we finally determine a set of simple, rapid, and high-synchronous rate synchronization method suitable for simultaneous treatment of HepG2and L02, including synchronization at Gl phase by one thymidine block, synchronization at S and G2phases by double thymidine block, and synchronization at M phase by nocodazole block and collection by gentle shaking. The flow cytometric analysis of cell cycle showed that we can obtain (86.64±1.33)%,(74.28±1.06)%,(91.26±1.62)%and (81.93±1.37)%for synchronized HepG2at G1, S, G2and M phase, respectively; and (73.44±1.43)%,(75.37±1.42)%,(73.14±0.96)%and (72.06±1.24)%for synchronized L02, respectively, by using the eatablished synchronization methods. And when compared with control group, the synchronized cells after the drug treatment showed significant difference (P<0.05), which can meet the demand of subsequent lectin microarray analysis of glycoprotein at specific phase of cell cycle.Part2:The analysis of glycan structural changes of glycoprotein at different phases of cell cycle by using lectin microarray technologyIn this part, the established lectin microarray method in our laboratory was used to analyze the total proteins of synchronized HepG2and L02at G1, S, G2and M phase, and the microarray data were normalized by median-normalized method. According to the three categories that HepG2cell cycle progression, L02cell cycle progression and comparison of HepG2with L02at different phases of cell cycle, we comparatively analyze glycan structures of the involved glycoproteins. By screening the lectins that have bonding strength differences in cell cycle, we can infer the differential expression of glycan structures on the glycoproteins of the two cell lines during different phases of cell cycle based on the binding specificities between the lectins and glycans:(1) The differential expression of glycan structures on the glycoproteins in the cell cycle progression of HepG2, for example, glycan structures of poly-al-3/al-6Man, poly-LacNAc and (GlcNAc)n, terminal GalNAc, GalNAcα-Ser/Thr(Tn-antigen), bisecting GlcNAc, biantennary N-glycan and tetraantennary complex-type N-glycan are in low expression in G1phase, while high expression in S, G2and M phases.(2) The differential expression of glycan structures on the glycoproteins in the cell cycle progression of L02, for example, glycan structures of Galβ1-3GalNAca-Ser/Thr(T-antigen) and GalNAcα-Ser/Thr(Tn-antigen), GlcNAc and galactosylated N-glycans, branched and terminal mannose and terminal GlcNAc, and Siaa2-3Ga1β1-4GlcNAc and Ga1β1-4GlcNAc are in low expression in G1phase, while high expression in S, G2and M phases. And then by comparing the bonding strength differences of lectins in each phase of cell cycle, we also can infer that there are seven categories of characteristic changes in glycan structure during cell cycle of HepG2, including:(1) The glycan structures of GlcNAc and galactosylated N-glycans, branched and terminal mannose and terminal GlcNAc, and terminala-1,3mannose are in high expression in G1phase, while low expression in S phase;(2) The glycan structures of terminalaFuc and Sia-Lex are in high expression in G1phase, while low expression in G2phase;(4) The glycan structures of poly-al-3/al-6Man, and a-Gal and a-GalNAc are only high expression in S phase;(5) The glycan structures of terminal GalNAc, poly-LacNAc and (GlcNAc)n, and β-gal are only low expression in G1phase;(6) The glycan structures of>Biantennary,(GlcNAc)n, poly-LacNAc and LacNAc, and bisecting GlcNAc and biantennary N-glycans and tetraantennary complex-type N-glycan are in low expression in G1phase, while high expression in S phase;(7) The glycan structures of Ga1β1-3GalNAcα-Ser/Thr(T-antigen), GalNAcα-Ser/Thr(Tn-antigen), and Fucal-6GlcNAc(core fucose) are in low expression in G1phase. The results suggested that we can predict the occurrence of HCC at an earlier stage and discover several potential glycan markers for early diagnosis of HCC by analysis of the structural differences of these glycans in cell cycle.Part3:Validation of the lectin microarray results by using lectin histochemistryAccording to the obtained lectins that have bonding strength differences in different phases from the microarray comparative analysis of HepG2with L02, seven representative difference lectins (GSL-Ⅱ、AAL、GNA、DSA、PHA-E+L Jacalin and PSA) and one lectin that there is no difference variation in four phases of HepG2and L02(UEA-Ⅰ) were selected to analyze the binding differences of synchronized HepG2and L02at G1, S and G2phase by lectin histochemistry. The results of lectin histochemistry showed that the selected lectins can bind specifically to different cell regions and show different bonding strengths, which fully agreed with the results obtained by the lectin microarrays. At the same time, the results can assess the binding region of each lectin in the cells of different phases, which can facilitate further understanding of the biological function of the glycan molecules.In summary, we constructed the characteristic changes of the glycoprotein glycopatterns in cell cycle progression of human hepatoma cell. Through analysis of the changes of glycan structures in cell cycle progression of human hepatoma cell, it can provide the basis for discovering new glycan markers for diagnosis of HCC at an earlier stage, and provide a new potential target for the diagnosis and treatment of HCC. |
PDF Full Text Request