α-1,2甘露醣酶是N-醣基化的主要酵素，其參與真核生物中醣蛋白的形成。目前研究發現α-1,2甘露醣酶的調控異常會導致癌症的發生，然而確切的機轉仍不明。在我們的研究中發現α-1,2甘露醣酶I的四個亞型：α-1,2甘露醣酶IA1、α-1,2甘露醣酶IB1、α-1,2甘露醣酶IA2及α-1,2甘露醣酶IC1分別在肝癌形成的過程中扮演不同的角色。從臨床病理特性分析發現：A1、B1及A2三個亞型的高度表達與臨床階段、腫瘤大小、甲型蛋白含量及侵襲程度呈正相關；而C1亞型，其表達量在肝癌初期極低的現象與另外三者截然不同。依據臨床病理分析結果進而發現若是肝癌病人的A1、B1及A2亞型過度表達伴隨著C1亞型低表現，其五年存活率有明顯降低的情形發生。因此從α-1,2甘露醣酶I亞型的功能分析發現：α-1,2甘露醣酶IA1的過量表達顯著的提升細胞增生、遷移、轉形能力及細胞在斑馬魚體內的遷移能力；然而α-1,2甘露醣酶IC1的過量表達展現相反的現象並且造成細胞週期停滯。更進一步針對引發細胞現象變化的相關基因群觀察，其表達量也隨著上升（α-1,2甘露醣酶IA1）或降低（α-1,2甘露醣酶IC1），同時發現α-1,2甘露醣酶IA1的過量表達所誘發上述的細胞現象改變，是由於活化未折疊蛋白反應路徑的主要調控因子。最後在我們所建立的肝臟特異性表達α-1,2甘露醣酶IA1與α-1,2甘露醣酶IC1轉殖基因斑馬魚動物模式中證實，α-1,2甘露醣酶IA1是一個高潛力致癌基因，其高度表達會誘發肝癌產生藉由活化未折疊蛋白路徑反應；而α-1,2甘露醣酶IC1在肝癌癌化過程中的高度表達可能具備了抑制腫瘤的能力。

α-1,2 mannosidases, key enzymes in N-glycosylation, are required for the formation of mature glycoproteins in eukaryotes. Aberrant regulation of α-1,2 mannosidases can result in cancer although the underlying mechanisms are unclear. Here we report the distinct roles of α-1,2 mannosidase subtypes in the formation of hepatocellular carcinoma (HCC). Clinicopathological analyses revealed that the clinical stage, tumor size, α-fetoprotein level and invasion status were positively correlated with the expression levels of MAN1A1, MAN1B1, and MAN1A2. In contrast, the expression of MAN1C1 was decreased as early as stage I of HCC. Survival analyses showed that high MAN1A1, MAN1A2, and MAN1B1 expression levels, combined with low MAN1C1 expression levels, were significantly correlated with shorter overall survival rate. Functionally, the overexpression of MAN1A1 promoted proliferation, migration, and transformation as well as in vivo migration in zebrafish. Conversely, overexpression of MAN1C1 reduced the migration ability both in vitro and in vivo, decreased the colony formation ability, and shortened the S phase of the cell cycle. Furthermore, the expression of genes involved in cell cycle/proliferation- and migration was increased in MAN1A1-overexpressing cells but decreased in MAN1C1-overexpressing cells. Besides, MAN1A1 activated the expression of key regulators of the unfolded protein response while treatment with ER stress inhibitors blocked the expression of MAN1A1-activated genes. Consequently, using the MAN1A1 liver-specific overexpression zebrafish model, we observed steatosis and inflammation at earlier stages and HCC formation at a later stage accompanied by the increased expression of the UPR modulator, BiP. These data suggest that the up-regulation of MAN1A1 activates UPR and might initiates metastasis. Together our study demonstrates that MAN1A1 represents a novel oncogene while MAN1C1 plays a role in tumor suppression in hepatocarcinogenesis.

Abstract............................................................................................................................... I
中文摘要.............................................................................................................................II
誌謝................................................................................................................................... III
目錄................................................................................................................................... IV Chapter 1 Introduction......................................................................................................1
1.1 Liver diseases and Hepatocellular carcinoma............................................................1
1.2 Discovery of Class I α-1, 2 mannosidases as biomarkers for HCC...........................2
1.3 The function of α-1, 2-mannosidases.........................................................................2
1.4 Dysregulation of α-1,2 mannosidases participated in cancer progression.................4
1.5 α-1,2 mannosidases represent novel biomarkers in HCC..........................................5
Chapter 2 Materials and Methods....................................................................................7
2.1 Human HCC samples.................................................................................................7
2.2 Cell culture.................................................................................................................7
2.3 Plasmid and reagents..................................................................................................7
2.3.1 Constructs for functional assay...........................................................................7
2.3.2 Constructs for transgenic fish .............................................................................8
2.4 Transfection and reagents ..........................................................................................9
2.4.1 Transient transfection..........................................................................................9
2.4.2 Selection for stably expression clone..................................................................9
2.4.3 shRNA knockdown...........................................................................................10
2.5 Functional assay.......................................................................................................10
2.5.1 In vitro Migration Assay...................................................................................10
2.5.2 Colony Formation Assay ..................................................................................10
2.5.3 Cell Proliferation Assay....................................................................................11
2.5.4 Flow Cytometry ................................................................................................11
2.6 Analysis for RNA expression level..........................................................................12
2.6.1 RNA extraction .................................................................................................12
2.6.2 Reverse-transcription Polymerase Chain Reaction (RT-PCR).........................12
2.6.3 Quantitative Polymerase Chain Reaction (Q-PCR)..........................................12
2.7 Zebrafish Husbandary..............................................................................................13
2.8 Xenotransplantation and image analysis..................................................................13
2.9 Histopathological Staining.......................................................................................14
2.9.1 Immunohistochemistry staining........................................................................14
2.9.2 Oil Red O staining ............................................................................................15
2.9.3 Sirius Red O staining ........................................................................................15
2.10 SDS-PAGE and Western Blotting .........................................................................16

2.11 Statistical Analysis.................................................................................................16
Chapter 3 Results.............................................................................................................17
3.1 Differential Expression of α-1, 2-mannosidase I Subtypes in Human HCC Tissues.................................................................................................................... 17
3.2 Determined cell lines for functional assay...............................................................18
3.3 In In Vitro Assays, Overexpression of MAN1A1 Increases Cell Migration Ability While MAN1C1 Overexpression Decreases Cell Migration..................................19
3.4 Utilized xenotransplantation to observe metastasis in vivo .....................................20
3.5 In Colony Formation Assays, the Overexpression of MAN1A1 and MAN1C1
have Opposite Effects on Cellular Transformation ...............................................21
3.6 Overexpression of MAN1A1 Increases Cell Proliferation While Overexpression
of MAN1C1 Leads to Cell Cycle Arrest ................................................................22
3.7 MAN1A1 and MAN1C1 Have Opposite Effects on MMP9, PCNA, and CCNA2 Expression in Cell Migration, Proliferation, and Cell Cycle Regulation ..............22
3.8 UPR Regulators are Activated in MAN1A1-Overexpressing Cells and Repressed
in MAN1C1-Overexpressing Cells.........................................................................23
3.9 ER stress inhibitors revised the expression of MAN1A1-induced genes .................24
3.10 Overexpression of MAN1A1 Induces Hepatocarcinogenesis in Zebrafish...........25
3.11 Treatment with a Mannosidase Inhibitor, ER Stress Inhibitors, or MAN1A1 shRNA Reduced MAN1A1-Overexpression-Mediated Cell Proliferation in the Xenotransplantation Assay ....................................................................................26
Chapter 4 Discussion .......................................................................................................28
4.1 The expression patterns of α-1,2 mannosidases.......................................................28
4.2 The activation of UPR during hepatocarcinogenesis...............................................28
4.3 The possible mechanism of ER stress induction......................................................29
4.4 Conclusion ...............................................................................................................30
4.5 Future perspective....................................................................................................32
List of Tables ....................................................................................................................34
Table 1 primers and shRNA target sequences ...............................................................34
Primer for PCR cloning: ............................................................................................34
Primers for sequencing ..............................................................................................34
shRNA target sequence..............................................................................................34
Primers for Gateway cloning .....................................................................................35
Primers for real-time PCR (Human gene) .................................................................35
Primers for real-time PCR (Zebrafish gene)..............................................................36
Table 2. Summary of the Clinicopathological Correlation with MAN1A1 Expression Levels in the HBV(+)-HCC Patients .....................................................................37
V
Table 3. Summary of the Clinicopathological Correlation with MAN1A2 Expression Levels in the HBV(+)-HCC Patients .....................................................................38
Table 4. Summary of the Clinicopathological Correlation with MAN1B1 Expression Levels in the HBV(+)-HCC Patients .....................................................................39
Table 5. Summary of the Clinicopathological Correlation with MAN1C1 Expression Levels in the HBV(+)-HCC Patients .....................................................................40
List of Figures and Figure Legends................................................................................41
Figure 1. The mRNA Level of the Four Human MAN1 Genes in Different Stages of Hepatocellular Carcinoma. ....................................................................................41
Figure 2. The mRNA Level of the Four Human MAN1 Genes in Hepatoma Cell Lines.......................................................................................................................43
Figure 3. The Protein Expression Patterns of the MAN1A1 and MAN1C1 in
Different Stages of Hepatocarcinogenesis.............................................................45
Figure 4. Statistical Analysis of the Protein Levels of MAN1A1 and MAN1C1
Showed an Upregulation of MAN1A1 and Downregulation of MAN1C1
During Hepatocarcinogenesis. ...............................................................................47
Figure 5. Survival Curve Analysis Showed High Level of MAN1A2, A2, B1 and Low Level of MAN1C1 Correlated to Poor Overall Survival........................................49
Figure 6 In vivo Xenotransplantation Assay for Different Cell Lines. .......................... 51
Figure 7. In Vitro Migration Assay of PLC5 and Hep3B and Selection of Cells..........53
Figure 8. Transient overexpression of MAN1A1 Enhances, and MAN1C1 Decreases
the Migration Ability of Cells In Vitro. .................................................................55
Figure 9. shRNA knockdown of MAN1A1, MAN1A2, and MAN1B1 Decreases the Migration Ability of Cells In Vitro. .......................................................................57
Figure 10. shRNA Knockdown of MAN1C1 Increased the Migration Ability of Cells
In Vitro...................................................................................................................59
Figure 11. Stable Overexpression of MAN1A1 Enhances, and MAN1C1 Decreases
the Migration Ability of Cells In Vitro. .................................................................61
Figure 12. In Vivo Xenotransplantation Assay for MAN1A1-Overexpressing Cells labeled by DiI.........................................................................................................63
Figure 13. In Vivo Xenotransplantation Assay for MAN1C1-Overexpressing Cells labeled by DiI.........................................................................................................65
Figure 14. In Vivo Xenotransplantation Assay for MAN1A1-Overexpressing Cells labeled by CFSE. ...................................................................................................67
Figure 15. In Vivo Xenotransplantation Assay for MAN1C1-Overexpressing Cells labeled by CFSE. ...................................................................................................69
Figure 16. In Vivo Proliferation Assay for MAN1A1-Overexpressing Cells labeled by CFSE. ..................................................................................................................... 71
VI
Figure 17. In Vivo Proliferation Assay for MAN1C1-Overexpressing Cells labeled by CFSE. ..................................................................................................................... 73
Figure 18. Colony Formation of MAN1A1 or MAN1C1 Overexpressing Cell Lines. ...75
Figure 19. Proliferation Ability of MAN1A1 or MAN1C1 Overexpressing Cell Lines. 78
Figure 20. Cell Cycle of MAN1C1 Overexpressing Cell Lines.....................................80
Figure 21. Expression Patterns of MMP9, CCNA2, PCNA and UPR Regulator in MAN1A1 or MAN1C1 Overexpressing Stable Cell Lines......................................82
Figure 22. MAN1A1 Overexpression Activates the UPR and the Expression of Downstream Genes. ...............................................................................................86
Figure 23. Histopathological Analysis of Hepatocytes in MAN1A1 and MAN1C1 Transgenic Fish at 3, 5, 7, 9 and 11 Months of Age...............................................91
Figure 24. Symptoms of MAN1A1 and MAN1C1 Transgenic Fish at 3, 5, 7, 9 and
11 Months of Age...................................................................................................95
Figure 25. Examination of the Progression of Hepatocarcinogenesis in MAN1A1
and MAN1C1 Overexpressing Transgenic and Control Fish by Oil Red O and Sirius Red Staining. ...............................................................................................97
Figure 26. Quantifications of Fibrosis Level in MAN1A1 Overexpressing, MAN1C1 Overexpressing Transgenic and control Fish by Sirius Red Staining....................99
Figure 27. Q-PCR Analysis of the Expression of Lipogenic Factors in Zebrafish Overexpressing MAN1A1 or MAN1C1 in the Liver. .........................................101
Figure 28. Q-PCR Analysis of the Expression of Cell Cycle Related Genes in
Zebrafish Overexpressing MAN1A1 or MAN1C1 in the Liver..........................103
Figure 29. Q-PCR Analysis of the Expression of ER Stress Related Genes in
Zebrafish Overexpressing MAN1A1 or MAN1C1 in the Liver..........................105
Figure 30. Examination of the Expression of the UPR mediator BiP in Transgenic
Fish Overexpressing MAN1A1 and MAN1C1 by IHC Staining. .......................107
Figure 31. In Vivo Xenotransplantation Assay for Different Inhibitors or MAN1A1 shRNA Treatment in MAN1A1-Overexpressing Cells. .......................................109 References.......................................................................................................................111 Appendixes...................................................................................................................... 129
Appendix 1: The expression level of α-1, 2 mannosidases in HBx induced HCC
mouse ................................................................................................................... 129
Appendix 2: Protein sequencing of MAN1A1.............................................................130
Appendix 3: protein sequencing of MAN1C1.............................................................131
Appendix 4: Tg(fabp10a:MAN1A1)(up) & Tg(fabp10a:MAN1C1)(down) at 2dpf...132
Appendix 5: the expression vector used in functional assay (picture from Clontech) 133
Appendix 6: protein expression in normal tissues and cancer cell lines for
MAN1A1 .............................................................................................................134
Appendix 7: protein expression in normal tissues and cancer cell lines for
MAN1A2 .............................................................................................................136
Appendix 8: protein expression in normal tissues and cancer cell lines for MAN1B1..............................................................................................................138
Appendix 9: protein expression in normal tissues and cancer cell lines for MAN1C1.............................................................................................................. 140
Appendix 10: ERSE and NF-kB binding site in the downstream target genes that
might be activated by MAN1A1..........................................................................142
Appendix 11: Dysregulation of α-1, 2 mannosidases I promoted proliferation and migration ability via UPR ....................................................................................143
Appendix 12: Comparative glycomic mapping in Hep3B, MAN1A1/Hep3B and MAN1A1 shRNA/Hep3B....................................................................................144
Appendix 13: Comparative glycomic mapping in 293T, MAN1A1/293T and
MAN1A1 shRNA/293T.......................................................................................145
Appendix 14: Comparative glycomic mapping in Hep3B and MAN1C1/Hep3B ......146
Publications ....................................................................................................................147 Awards ............................................................................................................................148

Anisimov, V.N., 2007. Biology of aging and cancer. Cancer Control 14, 23-31.
Baffy, G., Brunt, E.M., Caldwell, S.H., 2012. Hepatocellular carcinoma in non-alcoholic fatty liver disease: an emerging menace. J Hepatol 56, 1384-1391.
Barlowe, C.K., Miller, E.A., 2013. Secretory protein biogenesis and traffic in the early secretory pathway. Genetics 193, 383-410.
Begum, J., Day, W., Henderson, C., Purewal, S., Cerveira, J., Summers, H., Rees, P., Davies, D., Filby, A., 2013. A method for evaluating the use of fluorescent dyes to track proliferation in cell lines by dye dilution. Cytometry A 83, 1085-1095.
Benson, A.B., 3rd, Abrams, T.A., Ben-Josef, E., Bloomston, P.M., Botha, J.F., Clary, B.M., Covey, A., Curley, S.A., D'Angelica, M.I., Davila, R., Ensminger, W.D., Gibbs, J.F., Laheru, D., Malafa, M.P., Marrero, J., Meranze, S.G., Mulvihill, S.J., Park, J.O., Posey, J.A., Sachdev, J., Salem, R., Sigurdson, E.R., Sofocleous, C., Vauthey, J.N., Venook, A.P., Goff, L.W., Yen, Y., Zhu, A.X., 2009. NCCN clinical practice guidelines in oncology: hepatobiliary cancers. J Natl Compr Canc Netw 7, 350-391.
Bergers, G., Benjamin, L.E., 2003. Tumorigenesis and the angiogenic switch. Nature
111
reviews. Cancer 3, 401-410.
Blagosklonny, M.V., 2008. Prevention of cancer by inhibiting aging. Cancer biology & therapy 7, 1520-1524.
Campisi, J., 2013. Aging, cellular senescence, and cancer. Annu Rev Physiol 75, 685-705.
Chen, C.J., Yu, M.W., Liaw, Y.F., 1997. Epidemiological characteristics and risk factors of hepatocellular carcinoma. J Gastroenterol Hepatol 12, S294-308.
Ciou, S.C., Chou, Y.T., Liu, Y.L., Nieh, Y.C., Lu, J.W., Huang, S.F., Chou, Y.T., Cheng, L.H., Lo, J.F., Chen, M.J., Yang, M.C., Yuh, C.H., Wang, H.D., 2015. Ribose-5-phosphate isomerase A regulates hepatocarcinogenesis via PP2A and ERK signaling. International journal of cancer. Journal international du cancer 137, 104-115.
Clark, J.M., Diehl, A.M., 2003. Nonalcoholic fatty liver disease: an underrecognized cause of cryptogenic cirrhosis. JAMA 289, 3000-3004.
Dai, R., Chen, R., Li, H., 2009. Cross-talk between PI3K/Akt and MEK/ERK pathways mediates endoplasmic reticulum stress-induced cell cycle progression and cell death in human hepatocellular carcinoma cells. Int J Oncol 34, 1749-1757.
112
de Almeida, S.F., Picarote, G., Fleming, J.V., Carmo-Fonseca, M., Azevedo, J.E., de Sousa, M., 2007. Chemical chaperones reduce endoplasmic reticulum stress and prevent mutant HFE aggregate formation. J Biol Chem 282, 27905-27912.
Dennis, J.W., Granovsky, M., Warren, C.E., 1999. Glycoprotein glycosylation and cancer progression. Biochim Biophys Acta 1473, 21-34.
Dennis, J.W., White, S.L., Freer, A.M., Dime, D., 1993. Carbonoyloxy analogs of the anti-metastatic drug swainsonine. Activation in tumor cells by esterases. Biochemical pharmacology 46, 1459-1466.
Diaz-Gonzalez, A., Forner, A., 2016. Surveillance for hepatocellular carcinoma. Best Pract Res Clin Gastroenterol 30, 1001-1010.
Egeblad, M., Werb, Z., 2002. New functions for the matrix metalloproteinases in cancer progression. Nature reviews. Cancer 2, 161-174.
El-Serag, H.B., 2012. Epidemiology of viral hepatitis and hepatocellular carcinoma. Gastroenterology 142, 1264-1273 e1261.
European Association For The Study Of The, L., European Organisation For, R.,
113
Treatment Of, C., 2012. EASL-EORTC clinical practice guidelines: management of hepatocellular carcinoma. J Hepatol 56, 908-943.
Falandry, C., Bonnefoy, M., Freyer, G., Gilson, E., 2014. Biology of cancer and aging: a complex association with cellular senescence. J Clin Oncol 32, 2604-2610.
Farazi, P.A., DePinho, R.A., 2006. Hepatocellular carcinoma pathogenesis: from genes to environment. Nat Rev Cancer 6, 674-687.
Ferlay, J., Shin, H.R., Bray, F., Forman, D., Mathers, C., Parkin, D.M., 2010. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. International journal of cancer. Journal international du cancer 127, 2893-2917.
Fulcher, D., Wong, S., 1999. Carboxyfluorescein succinimidyl ester-based proliferative assays for assessment of T cell function in the diagnostic laboratory. Immunol Cell Biol 77, 559-564.
Gerber-Lemaire, S., Juillerat-Jeanneret, L., 2010. Studies toward new anti-cancer strategies based on alpha-mannosidase inhibition. Chimia 64, 634-639.
Gjymishka, A., Su, N., Kilberg, M.S., 2009. Transcriptional induction of the human
114
asparagine synthetase gene during the unfolded protein response does not require the ATF6 and IRE1/XBP1 arms of the pathway. Biochem J 417, 695-703.
Goss, P.E., Baker, M.A., Carver, J.P., Dennis, J.W., 1995. Inhibitors of carbohydrate processing: A new class of anticancer agents. Clin Cancer Res 1, 935-944.
Hakomori, S., 1996. Tumor malignancy defined by aberrant glycosylation and sphingo(glyco)lipid metabolism. Cancer Res 56, 5309-5318.
Hazari, Y.M., Bashir, A., Haq, E.U., Fazili, K.M., 2016. Emerging tale of UPR and cancer: an essentiality for malignancy. Tumour Biol.
Herscovics, A., 2001. Structure and function of Class I alpha 1,2-mannosidases involved in glycoprotein synthesis and endoplasmic reticulum quality control. Biochimie 83, 757- 762.
Hori, H., Suzuki, M., Inagaki, H., Oshima, T., Koga, A., 1998. An active Ac-like transposable element in teleost fish. J Mar Biotechnol 6, 206-207.
Hosokawa, N., You, Z., Tremblay, L.O., Nagata, K., Herscovics, A., 2007. Stimulation of ERAD of misfolded null Hong Kong alpha1-antitrypsin by Golgi alpha1,2-mannosidases. 115

Biochemical and biophysical research communications 362, 626-632.
Hotamisligil, G.S., 2010. Endoplasmic reticulum stress and the inflammatory basis of metabolic disease. Cell 140, 900-917.
Hung, J.H., Su, I.J., Lei, H.Y., Wang, H.C., Lin, W.C., Chang, W.T., Huang, W., Chang, W.C., Chang, Y.S., Chen, C.C., Lai, M.D., 2004. Endoplasmic reticulum stress stimulates the expression of cyclooxygenase-2 through activation of NF-kappaB and pp38 mitogen- activated protein kinase. J Biol Chem 279, 46384-46392.
Kawakami, K., 2004. Transgenesis and gene trap methods in zebrafish by using the Tol2 transposable element. Methods Cell Biol 77, 201-222.
Kawakami, K., Koga, A., Hori, H., Shima, A., 1998. Excision of the tol2 transposable element of the medaka fish, Oryzias latipes, in zebrafish, Danio rerio. Gene 225, 17-22.
Kawakami, K., Shima, A., 1999. Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio. Gene 240, 239-244.
Kew, M.C., 2013. Aflatoxins as a cause of hepatocellular carcinoma. J Gastrointestin
116
Liver Dis 22, 305-310.
Kim, S.J., Sohn, I., Do, I.G., Jung, S.H., Ko, Y.H., Yoo, H.Y., Paik, S., Kim, W.S., 2014. Gene expression profiles for the prediction of progression-free survival in diffuse large B cell lymphoma: results of a DASL assay. Annals of hematology 93, 437-447.
Koike, K., Shirakata, Y., Yaginuma, K., Arii, M., Takada, S., Nakamura, I., Hayashi, Y., Kawada, M., Kobayashi, M., 1989. Oncogenic potential of hepatitis B virus. Mol Biol Med 6, 151-160.
Lai, Q., Vitale, A., Iesari, S., Finkenstedt, A., Mennini, G., Spoletini, G., Hoppe- Lotichius, M., Vennarecci, G., Manzia, T.M., Nicolini, D., Avolio, A.W., Frigo, A.C., Cillo, U., Graziadei, I., Rossi, M., Tsochatzis, E., Otto, G., Ettorre, G.M., Tisone, G., Vivarelli, M., Agnes, S., Lerut, J., European Hepatocellular Cancer Liver Transplant Study, G., 2017. Intention-to-treat survival benefit of liver transplantation in patients with hepatocellular cancer. Hepatology.
Leal, J.N., Kingham, T.P., 2015. Hepatic artery infusion chemotherapy for liver malignancy. Surg Oncol Clin N Am 24, 121-148.
Lee, A.H., Scapa, E.F., Cohen, D.E., Glimcher, L.H., 2008. Regulation of hepatic
117
lipogenesis by the transcription factor XBP1. Science 320, 1492-1496.
Lee, J.T., Herlyn, M., 2006. Embryogenesis meets tumorigenesis. Nat Med 12, 882-884.
Liu, T., Zhang, S., Chen, J., Jiang, K., Zhang, Q., Guo, K., Liu, Y., 2014. The transcriptional profiling of glycogenes associated with hepatocellular carcinoma metastasis. PLoS One 9, e107941.
Liu, Y., Ren, S., Xie, L., Cui, C., Xing, Y., Liu, C., Cao, B., Yang, F., Li, Y., Chen, X., Wei, Y., Lu, H., Jiang, J., 2015. Mutation of N-linked glycosylation at Asn548 in CD133 decreases its ability to promote hepatoma cell growth. Oncotarget 6, 20650-20660.
Liu, Y.L., Lu, W.C., Brummel, T.J., Yuh, C.H., Lin, P.T., Kao, T.Y., Li, F.Y., Liao, P.C., Benzer, S., Wang, H.D., 2009. Reduced expression of alpha-1,2-mannosidase I extends lifespan in Drosophila melanogaster and Caenorhabditis elegans. Aging Cell 8, 370-379.
Lu, J.W., Hsia, Y., Yang, W.Y., Lin, Y.I., Li, C.C., Tsai, T.F., Chang, K.W., Shieh, G.S., Tsai, S.F., Wang, H.D., Yuh, C.H., 2012. Identification of the common regulators for hepatocellular carcinoma induced by hepatitis B virus X antigen in a mouse model. Carcinogenesis 33, 209-219.
118
Lu, J.W., Liao, C.Y., Yang, W.Y., Lin, Y.M., Jin, S.L., Wang, H.D., Yuh, C.H., 2014. Overexpression of endothelin 1 triggers hepatocarcinogenesis in zebrafish and promotes cell proliferation and migration through the AKT pathway. PloS one 9, e85318.
Lu, J.W., Yang, W.Y., Lin, Y.M., Jin, S.L., Yuh, C.H., 2013a. Hepatitis B virus X antigen and aflatoxin B1 synergistically cause hepatitis, steatosis and liver hyperplasia in transgenic zebrafish. Acta histochemica 115, 728-739.
Lu, J.W., Yang, W.Y., Tsai, S.M., Lin, Y.M., Chang, P.H., Chen, J.R., Wang, H.D., Wu, J.L., Jin, S.L., Yuh, C.H., 2013b. Liver-specific expressions of HBx and src in the p53 mutant trigger hepatocarcinogenesis in zebrafish. PloS one 8, e76951.
Lu, W., Hagiwara, D., Morishita, Y., Tochiya, M., Azuma, Y., Suga, H., Goto, M., Banno, R., Sugimura, Y., Oyadomari, S., Mori, K., Arima, H., 2016. Unfolded protein response in hypothalamic cultures of wild-type and ATF6alpha-knockout mice. Neuroscience letters 612, 199-203.
Lu, Y., Xu, Y.Y., Fan, K.Y., Shen, Z.H., 2006. 1-Deoxymannojirimycin, the alpha1,2- mannosidase inhibitor, induced cellular endoplasmic reticulum stress in human hepatocarcinoma cell 7721. Biochem Biophys Res Commun 344, 221-225.
119
Marques, I.J., Weiss, F.U., Vlecken, D.H., Nitsche, C., Bakkers, J., Lagendijk, A.K., Partecke, L.I., Heidecke, C.D., Lerch, M.M., Bagowski, C.P., 2009. Metastatic behaviour of primary human tumours in a zebrafish xenotransplantation model. BMC cancer 9, 128.
Mazzanti, R., Arena, U., Tassi, R., 2016. Hepatocellular carcinoma: Where are we? World journal of experimental medicine 6, 21-36.
McDermott, S., Gervais, D.A., 2013. Radiofrequency ablation of liver tumors. Semin Intervent Radiol 30, 49-55.
McGuire, S., 2016. World Cancer Report 2014. Geneva, Switzerland: World Health Organization, International Agency for Research on Cancer, WHO Press, 2015. Advances in nutrition 7, 418-419.
Melo, A.P.S., Franca, E.B., Malta, D.C., Garcia, L.P., Mooney, M., Naghavi, M., 2017. Mortality due to cirrhosis, liver cancer, and disorders attributed to alcohol use: Global Burden of Disease in Brazil, 1990 and 2015. Rev Bras Epidemiol 20Suppl 01, 61-74.
Milde-Langosch, K., Karn, T., Schmidt, M., zu Eulenburg, C., Oliveira-Ferrer, L., Wirtz,
R.M., Schumacher, U., Witzel, I., Schutze, D., Muller, V., 2014. Prognostic relevance of
glycosylation-associated genes in breast cancer. Breast cancer research and treatment 145,
120
295-305.
Molinari, M., 2007. N-glycan structure dictates extension of protein folding or onset of disposal. Nat Chem Biol 3, 313-320.
Nicoli, S., Ribatti, D., Cotelli, F., Presta, M., 2007. Mammalian tumor xenografts induce neovascularization in zebrafish embryos. Cancer Res 67, 2927-2931.
Ogen-Shtern, N., Avezov, E., Shenkman, M., Benyair, R., Lederkremer, G.Z., 2016. Mannosidase IA is in Quality Control Vesicles and Participates in Glycoprotein Targeting to ERAD. Journal of molecular biology 428, 3194-3205.
Olszewska, E., Borzym-Kluczyk, M., Rzewnicki, I., Wojtowicz, J., Rogowski, M., Pietruski, J.K., Czajkowska, A., Sieskiewicz, A., 2012. Possible role of alpha- mannosidase and beta-galactosidase in larynx cancer. Contemp Oncol (Pozn) 16, 154- 158.
Oyadomari, S., Harding, H.P., Zhang, Y., Oyadomari, M., Ron, D., 2008. Dephosphorylation of translation initiation factor 2alpha enhances glucose tolerance and attenuates hepatosteatosis in mice. Cell metabolism 7, 520-532.
121
Ozcan, U., Yilmaz, E., Ozcan, L., Furuhashi, M., Vaillancourt, E., Smith, R.O., Gorgun, C.Z., Hotamisligil, G.S., 2006. Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes. Science 313, 1137-1140.
Pan, P.W., Zhang, Q., Hou, J., Liu, Z., Bai, F., Cao, M.R., Sun, T., Bai, G., 2012. Cell surface glycoprotein profiling of cancer cells based on bioorthogonal chemistry. Anal Bioanal Chem 403, 1661-1670.
Pan, S., Cheng, X., Chen, H., Castro, P.D., Ittmann, M.M., Hutson, A.W., Zapata, S.K., Sifers, R.N., 2013a. ERManI is a target of miR-125b and promotes transformation phenotypes in hepatocellular carcinoma (HCC). PloS one 8, e72829.
Pan, S., Cheng, X., Sifers, R.N., 2013b. Golgi-situated endoplasmic reticulum alpha-1, 2- mannosidase contributes to the retrieval of ERAD substrates through a direct interaction with gamma-COP. Molecular biology of the cell 24, 1111-1121.
Patel, P.S., Raval, G.N., Rawal, R.M., Patel, M.M., Balar, D.B., Patel, D.D., 1997. Importance of glycoproteins in human cancer. Indian J Biochem Biophys 34, 226-233.
Progatzky, F., Dallman, M.J., Lo Celso, C., 2013. From seeing to believing: labelling
strategies for in vivo cell-tracking experiments. Interface Focus 3, 20130001.
122
Quah, B.J., Parish, C.R., 2010. The use of carboxyfluorescein diacetate succinimidyl ester (CFSE) to monitor lymphocyte proliferation. J Vis Exp.
Rab, A., Bartoszewski, R., Jurkuvenaite, A., Wakefield, J., Collawn, J.F., Bebok, Z., 2007. Endoplasmic reticulum stress and the unfolded protein response regulate genomic cystic fibrosis transmembrane conductance regulator expression. Am J Physiol Cell Physiol 292, C756-766.
Ron, D., Walter, P., 2007. Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 8, 519-529.
Roth, J., Zuber, C., Park, S., Jang, I., Lee, Y., Kysela, K.G., Le Fourn, V., Santimaria, R., Guhl, B., Cho, J.W., 2010. Protein N-glycosylation, protein folding, and protein quality control. Mol Cells 30, 497-506.
Rouhi, P., Jensen, L.D., Cao, Z., Hosaka, K., Lanne, T., Wahlberg, E., Steffensen, J.F., Cao, Y., 2010. Hypoxia-induced metastasis model in embryonic zebrafish. Nat Protoc 5, 1911-1918.
Sapisochin, G., Bruix, J., 2017. Liver transplantation for hepatocellular carcinoma:
outcomes and novel surgical approaches. Nat Rev Gastroenterol Hepatol 14, 203-217.
123
Saraswat, V.A., Pandey, G., Shetty, S., 2014. Treatment algorithms for managing hepatocellular carcinoma. J Clin Exp Hepatol 4, S80-89.
Seifer, M., Hohne, M., Schaefer, S., Gerlich, W.H., 1991. In vitro tumorigenicity of hepatitis B virus DNA and HBx protein. J Hepatol 13 Suppl 4, S61-65.
Shuda, M., Kondoh, N., Imazeki, N., Tanaka, K., Okada, T., Mori, K., Hada, A., Arai, M., Wakatsuki, T., Matsubara, O., Yamamoto, N., Yamamoto, M., 2003. Activation of the ATF6, XBP1 and grp78 genes in human hepatocellular carcinoma: a possible involvement of the ER stress pathway in hepatocarcinogenesis. J Hepatol 38, 605-614.
Song, P.P., Xia, J.F., Inagaki, Y., Hasegawa, K., Sakamoto, Y., Kokudo, N., Tang, W., 2016. Controversies regarding and perspectives on clinical utility of biomarkers in hepatocellular carcinoma. World journal of gastroenterology 22, 262-274.
Sun, J.Y., Yang, H., Miao, S., Li, J.P., Wang, S.W., Zhu, M.Z., Xie, Y.H., Wang, J.B., Liu, Z., Yang, Q., 2009. Suppressive effects of swainsonine on C6 glioma cell in vitro and in vivo. Phytomedicine 16, 1070-1074.
Sun, J.Y., Zhu, M.Z., Wang, S.W., Miao, S., Xie, Y.H., Wang, J.B., 2007. Inhibition of the
growth of human gastric carcinoma in vivo and in vitro by swainsonine. Phytomedicine
124
14, 353-359.
Sun, S., Shi, G., Sha, H., Ji, Y., Han, X., Shu, X., Ma, H., Inoue, T., Gao, B., Kim, H., Bu, P., Guber, R.D., Shen, X., Lee, A.H., Iwawaki, T., Paton, A.W., Paton, J.C., Fang, D., Tsai, B., Yates, J.R., 3rd, Wu, H., Kersten, S., Long, Q., Duhamel, G.E., Simpson, K.W., Qi, L., 2015. IRE1alpha is an endogenous substrate of endoplasmic-reticulum-associated degradation. Nat Cell Biol 17, 1546-1555.
Tarantino, G., Saldalamacchia, G., Conca, P., Arena, A., 2007. Non-alcoholic fatty liver disease: further expression of the metabolic syndrome. J Gastroenterol Hepatol 22, 293- 303.
Taylor, A.M., Zon, L.I., 2009. Zebrafish tumor assays: the state of transplantation. Zebrafish 6, 339-346.
Tempel, W., Karaveg, K., Liu, Z.J., Rose, J., Wang, B.C., Moremen, K.W., 2004. Structure of mouse Golgi alpha-mannosidase IA reveals the molecular basis for substrate specificity among class 1 (family 47 glycosylhydrolase) alpha1,2-mannosidases. J Biol Chem 279, 29774-29786.
Tien, Y.W., Lee, P.H., Hu, R.H., Hsu, S.M., Chang, K.J., 2003. The role of gelatinase in
125
hepatic metastasis of colorectal cancer. Clin Cancer Res 9, 4891-4896.
Tseng, W.F., Jang, T.H., Huang, C.B., Yuh, C.H., 2011. An evolutionarily conserved kernel of gata5, gata6, otx2 and prdm1a operates in the formation of endoderm in zebrafish. Dev Biol 357, 541-557.
Vandewynckel, Y.P., Laukens, D., Devisscher, L., Paridaens, A., Bogaerts, E., Verhelst, X., Van den Bussche, A., Raevens, S., Van Steenkiste, C., Van Troys, M., Ampe, C., Descamps, B., Vanhove, C., Govaere, O., Geerts, A., Van Vlierberghe, H., 2015. Tauroursodeoxycholic acid dampens oncogenic apoptosis induced by endoplasmic reticulum stress during hepatocarcinogen exposure. Oncotarget 6, 28011-28025.
Vojta, A., Samarzija, I., Bockor, L., Zoldos, V., 2016. Glyco-genes change expression in cancer through aberrant methylation. Biochimica et biophysica acta.
Walter, P., Ron, D., 2011. The unfolded protein response: from stress pathway to homeostatic regulation. Science 334, 1081-1086.
Wang, M., Kaufman, R.J., 2016. Protein misfolding in the endoplasmic reticulum as a conduit to human disease. Nature 529, 326-335.
126
Wang, Q., Groenendyk, J., Michalak, M., 2015. Glycoprotein Quality Control and Endoplasmic Reticulum Stress. Molecules 20, 13689-13704.
Weinstein, B., 2002. Vascular cell biology in vivo: a new piscine paradigm? Trends Cell Biol 12, 439-445.
White, R.M., Sessa, A., Burke, C., Bowman, T., LeBlanc, J., Ceol, C., Bourque, C., Dovey, M., Goessling, W., Burns, C.E., Zon, L.I., 2008. Transparent adult zebrafish as a tool for in vivo transplantation analysis. Cell Stem Cell 2, 183-189.
Wu, H.C., Wang, Q., Yang, H.I., Ahsan, H., Tsai, W.Y., Wang, L.Y., Chen, S.Y., Chen, C.J., Santella, R.M., 2009. Aflatoxin B1 exposure, hepatitis B virus infection, and hepatocellular carcinoma in Taiwan. Cancer Epidemiol Biomarkers Prev 18, 846-853.
Yoshiuchi, K., Kaneto, H., Matsuoka, T.A., Kasami, R., Kohno, K., Iwawaki, T., Nakatani, Y., Yamasaki, Y., Shimomura, I., Matsuhisa, M., 2009. Pioglitazone reduces ER stress in the liver: direct monitoring of in vivo ER stress using ER stress-activated indicator transgenic mice. Endocrine journal 56, 1103-1111.
You, M.S., Jiang, Y.J., Yuh, C.H., Wang, C.M., Tang, C.H., Chuang, Y.J., Lin, B.H., Wu,
J.L., Hwang, S.P., 2016. A Sketch of the Taiwan Zebrafish Core Facility. Zebrafish 13
127
Suppl 1, S24-29.
Zhang, W.Y., Xu, F.Q., Shan, C.L., Xiang, R., Ye, L.H., Zhang, X.D., 2009. Gene expression profiles of human liver cells mediated by hepatitis B virus X protein. Acta Pharmacol Sin 30, 424-434.
Zhang, Y., Zhao, J.H., Zhang, X.Y., Guo, H.B., Liu, F., Chen, H.L., 2004. Relations of the type and branch of surface N-glycans to cell adhesion, migration and integrin expressions. Molecular and cellular biochemistry 260, 137-146.