肝細胞癌是世界上第五大常見癌症，主要好發於男性。肝癌的形成牽涉到多種病因與階段，流行病學研究顯示：B或C型肝炎病毒感染、脂肪肝、肝硬化、黃麴毒素的攝取與基因突變等都可能是導致肝癌的危險因子。甘胺酸氮甲基轉移酶 (簡稱GNMT)在正常肝臟中含量豐富，但是在肝細胞癌組織中的表現卻被抑制。過去研究指出GNMT能與環境致癌物 (多環芳香族碳氫化合物：benao[a]pyrene，簡稱BaP) 結合，並拮抗這些致癌物所產生的細胞毒性。為了進一步研究GNMT在肝癌的形成中所扮演的角色，我們實驗室建立一個GNMT基因剔除鼠模式，發現GNMT基因剔除會產生慢性肝炎與肝醣堆積。
在本研究中，我們持續追蹤這些小鼠長達兩年，並觀察到「自發性肝癌」的產生。100%的GNMT基因剔除母鼠有自發性的肝細胞癌產生；但只有二分之ㄧ的公鼠產生肝癌。我們也發現，GNMT基因剔除造成DNA甲基化程度的改變，也影響DNA甲基轉移酵素基因、致癌基因與抑癌基因的表現。在微陣列分析的研究中顯示，MAPK訊息傳導路徑在GNMT基因剔除母鼠的表現較高。利用二維凝膠電泳分析研究發現，11周齡的GNMT基因剔除鼠肝臟中，與解毒路徑與抗氧化功能相關的蛋白質表現降低，肝臟中的氧化壓力也較高。若將細胞大量表現GNMT，則能抑制過氧化物所產生的細胞毒性。此外，我們同時將野生型與GNMT基因剔除鼠施打BaP，來研究GNMT與環境致癌物之間的關係，結果顯示：GNMT基因剔除會藉由影響小鼠肝臟的解毒路徑與抗氧化功能，而使GNMT基因剔除鼠對於環境致癌物（BaP）的感受性變高，進而促使肝癌的發生。
除此之外，本研究也發現GNMT基因剔除鼠有高脂血症與脂肪肝等症狀。在人類脂肪肝組織與小鼠誘發脂肪肝模式均發現，GNMT在脂肪肝的表現顯著降低。為了進一步探討GNMT在脂肪代謝與脂肪肝形成過程中所扮演的角色，我們利用酵母菌雙雜合系統發現GNMT會與Niemann-Pick C2 (簡稱 NPC2) 蛋白結合。NPC2是一個膽固醇的結合蛋白，主要分布於溶小體，在膽固醇的運送及維持恆定上扮演重要角色。我們利用免疫沉澱法、間接免疫螢光染色與溶小體萃取實驗發現，GNMT在細胞質與NPC2有交互作用，並能延長NPC2蛋白的穩定度。進一步將低密度脂蛋白與黃體素處理細胞，結果發現當細胞大量表現GNMT時，能降低膽固醇累積在細胞內。此外，GNMT基因剔除鼠對缺乏甲硫胺酸與膽鹼（簡稱MCD）飼料所誘發產生的脂肪肝與肝損傷均較野生型小鼠來的嚴重，在本實驗中也發現，醣基化NPC2在脂肪肝形成過程中會顯著上升，有研究報導指出此醣基化NPC2會加速膽固醇的運輸速率。利用GNMT基因轉殖鼠餵食MCD飼料後顯示，GNMT會藉由增加醣基化NPC2蛋白的表現，而促進膽固醇運輸並減緩肝臟損傷與脂肪肝的形成。
綜合本研究的結果，我們推測GNMT除了透過影響肝臟甲基化程度、解毒功能與抗氧化能力，也會經由調節膽固醇代謝來維持肝細胞正常的功能。ㄧ旦GNMT的表現降低，就會導致細胞內許多訊息傳遞路徑失去平衡，進而促使肝癌發生。

Hepatocellular carcinoma (HCC) is the fifth common cancer in the world and its incidence in male is higher than in the women. The risk factors associated with HCC development are including chronic hepatitis virus infection (HBV and HCV), toxic exposure (alcohol consumption, aflatoxin and possibly smoking) and metabolic syndrome (non-alcoholic steatohepatitis). Glycine N-methyltransferase (GNMT) expresses abundantly in liver, but is down-regulated in HCC. Previously, we reported that GNMT interacts with benzo[a]pyrene (BaP) and alters the liver detoxification pathway and prevents DNA adducts formation and subsequent cytotoxicity. In order to clarify the role GNMT in the development of HCC, our lab generated a Gnmt knockout (Gnmt-/-) mouse model and showed that both genders of Gnmt-/- mice developed chronic hepatitis and glycogen storage disease.
In this study, we continuously followed up these Gnmt-/- mice to 24 months old and showed that all female and half of male Gnmt-/- mice developed HCC spontaneously. Global DNA hypomethylation and aberrant expression of oncogenes and tumor suppressor genes were involved in the tumorigenesis of Gnmt-/- mice. Microarray and real-time PCR analyses confirmed that mitogen-activated protein kinase (MAPK) pathway was activated in Gnmt-/- mice, especially in the female mice.
The two-dimensional gel electrophoresis coupled with mass spectrometry analyses also demonstrated that proteins involved in the detoxification and anti-oxidation response were significantly down-regulated in the 11 weeks of Gnmt-/- mice. In addition, lipid peroxidation was enhanced in Gnmt-/- mice. H2O2 treatment demonstrated that GNMT can antagonize H2O2-induced cytotoxic effects. Besides, BaP challenge experiments showed that Gnmt-/- mice is more susceptible to BaP-induced liver tumorigenesis.
In addition, loss of GNMT causes the progression of hyperlipidemia, steatosis and steatohepatitis before HCC development. In human and animal fatty liver tissues, the expression of GNMT is down-regulated. In order to elucidate the role of GNMT in the pathogenesis of steatosis and HCC, yeast two hybrid screening system was conducted and identified Niemann-Pick type C2 protein (NPC2) as a GNMT interacting protein. NPC2 is a lysosomal glycoprotein and involves in the regulating intracellular cholesterol homeostasis through direct binding with free cholesterol. Co-immunoprecipitation and immunofluorescence staining showed that NPC2 interacts and co-localizes with GNMT in cytosol. Low density lipoprotein and progesterone treatment demonstrated that NPC2 can release from lysosome into cytosol and co-localize with GNMT. In the meanwhile, GNMT doubled the half life of NPC2 isoforms and reduced cholesterol accumulation. In methionine and choline deficient (MCD) diet induced steatosis and steatohepatitis model, Gnmt-/- mice is more susceptible to MCD-induced steatosis and liver damage. On the contrary, Gnmt-transgenic mice attenuates MCD-induced liver damage and steatohepatitis through enhance glycosylated-NPC2. Accordingly, our data suggest that the development of hepatosteatosis may due to the effect of GNMT deficiency on the function of NPC2. Therefore, we proposed that the interaction between GNMT and NPC2 play an important role in the regulating of the pathogenesis for steatosis and HCC.
Taken together, multiple pathways may involve in the tumorigenesis of Gnmt-/- mice: (1) Global DNA hypomethylation and aberrant expression of oncogenes and tumor suppressor genes. (2) Down-regulation of detoxification and antioxidant-related proteins, which may trigger oxidative stress and increase the susceptibility of carcinogen (ex. BaP) induced liver damage and HCC. (3) Accumulate cholesterol in the hepatocytes and trigger the development of fatty liver through attenuate NPC2 protein stability.

Chinese Abstract-----------------------------------------1
English Abstract-----------------------------------------3
Contents-------------------------------------------------5
List of Figures------------------------------------------8
List of Tables------------------------------------------10
Chapter 1 General Introduction-------------------------11
1.1 Epidemiology of human hepatocellular carcinoma-----12
1.2 The mechanisms of steatosis to steatohepatitis (two-hits hypothesis)----------------------------------------15
1.3 Mouse models for steatohepatitis and HCC-----------17
1.4 Glycine N-methyltransferase (GNMT)-----------------22
1.5 Niemann-Pick Type C2 (NPC2)------------------------26
1.6 Objective and specific aims------------------------29

Chapter 2 Characterization of a glycine N-methyltransferase gene knockout mouse model for hepatocellular carcinoma---------------------------------30
2.1 Introduction----------------------------------------31
2.2 Materials andMethods--------------------------------31
2.2.1 Animal experiments--------------------------------31
2.2.2 Global DNA methylation----------------------------32
2.2.3 Dnmt activity-------------------------------------32
2.2.4 Microarray analysis and real-time PCR-------------32
2.3 Results---------------------------------------------33
2.3.1 The gender differences of HCC formation in Gnmt-/- mice-----------------------------------------------------33
2.3.2 Sexually dimorphic expression of Gnmt perturbs DNA methylation----------------------------------------------35
2.3.3 Aberrant expression of oncogenes and tumor suppressor in Gnmt-/- mice-------------------------------36
2.3.4 Microarray analysis of genes involved in various pathways at different stages of
tumorigenesis in Gnmt-/- mice-----------------------------36
2.3.5 MAPK pathway was activated in female Gnmt-/- mice--38
2.4 Discussion -------------------------------------- 38

Chapter 3 Deficiency of glycine N-methyltransferase results in deterioration of cellular defense to stress in mouse liver----------------------------------------------41
3.1 Introduction----------------------------------------42
3.2 Materials and Methods-------------------------------42
3.2.1 Sample preparation--------------------------------42
3.2.2 Two-DE gel electrophoresis, scanning and image analysis-------------------------------------------------43
3.2.3 In-gel digestion, mass spectrometry and database searching------------------------------------------------43
3.2.4 Real-time PCR and western blot--------------------45
3.2.5 Superoxide dismutase activity and lipid peroxidation----------------------------------------------------------45
3.2.6 H2O2 treatment and cytotoxicity assay-------------45
3.2.7 Benzo[a]pyrene treatment--------------------------46
3.3 Results---------------------------------------------46
3.3.1 Overall patterns of changes in proteomics analyses-46
3.3.2 Alteration of anti-oxidation, detoxification, one-carbon and glycolytic energy metabolism pathways in Gnmt-/- mice-----------------------------------------------------47
3.3.3 Liver damage and lipid peroxidation in Gnmt-/- mice-----------------------------------------------------------49
3.3.4 GNMT antagonizes H2O2-induced cytotoxic effects---------------------------------------------------------------49
3.3.5 Gross and pathological examination of BaP treated mice-----------------------------------------------------50
3.4 Discussion------------------------------------------51

Chapter 4 GNMT is involved in the intracellular trafficking of cholesterol through interacting with Niemann-Pick Disease Type C2 protein-----------------------------55
4.1 Introduction----------------------------------------55
4.2 Materials and Methods-------------------------------57
4.2.1 Mice maintenance and autopsy----------------------57
4.2.2 Serum and hepatic lipids measurement--------------57
4.2.3 Western blot and real-time PCR analysis-----------57
4.2.4 LDL and progesterone treatment--------------------58
4.2.5 Cycloheximide treatment---------------------------58
4.2.6 IHC staining of human fatty liver and HCC tissues-59
4.2.7 Statistical analyses------------------------------59
4.3 Results---------------------------------------------59
4.3.1 GNMT is down-regulated in human fatty liver and HCC tissues--------------------------------------------------59
4.3.2 GNMT is down-regulated in methionine and choline deficient diet induced hepatosteatosis model-------------60
4.3.3 Gnmt-/- mice develop hepatosteatosis and hyperlipidemia-------------------------------------------60
4.3.4 Cholesterol metabolism is impaired in the Gnmt-/- mice-----------------------------------------------------61
4.3.5 Identification of NPC2 as a GNMT binding protein--62
4.3.6 GNMT influences intracellular cholesterol trafficking through co-localizing with NPC2 in cytosol --63
4.3.7 GNMT enhances the stability of NPC2---------------64
4.3.8 GNMT attenuates MCD-induced steatohepatitis and liver damage through enhance glycosylated-NPC2-----------65
4.4 Discussion------------------------------------------66

Chapter 5 Conclusion------------------------------------68
Figures--------------------------------------------------69
Tables--------------------------------------------------114
References----------------------------------------------128
Appendices----------------------------------------------139

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