Lau Yee Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 147-157). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Abbreviations --- p.v / List of Figures --- p.viii / List of Tables --- p.xi / Contents --- p.xii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Hepatocellular carcinoma (HCC) --- p.1 / Chapter 1.1.1 --- Background of hepatocellular carcinoma (HCC) --- p.1 / Chapter 1.1.2 --- Etiology of HCC --- p.2 / Chapter 1.1.3 --- Relationship between HCC and HBV --- p.3 / Chapter 1.1.4 --- Differential gene expression under induction of HBx protein by microarray analysis --- p.5 / Chapter 1.1.5 --- Confirmation of candidate genes --- p.6 / Chapter 1.2 --- Ras-Oncogene --- p.8 / Chapter 1.2.1 --- Ras superfamily --- p.8 / Chapter 1.2.1.1 --- Rho family --- p.9 / Chapter 1.2.1.2 --- Rab family --- p.10 / Chapter 1.2.2 --- Functional mechanism of small GTPase --- p.11 / Chapter 1.2.3 --- Possible functions of Rho and Rab family members --- p.14 / Chapter 1.3 --- RhoC --- p.16 / Chapter 1.3.1 --- The genomic and protein structures of RhoC --- p.16 / Chapter 1.3.2 --- Relationship between RhoC and tumours --- p.19 / Chapter 1.4 --- Rabl4 --- p.20 / Chapter 1.4.1 --- The genomic and protein structures of Rabl4 --- p.20 / Chapter 1.4.2 --- Relationship between Rabl4 and tumours --- p.23 / Chapter 1.5 --- Aims of study --- p.23 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.25 / Chapter 2.1.1 --- Cell lines --- p.25 / Chapter 2.1.2 --- Cell culture reagents --- p.26 / Chapter 2.1.3 --- Reagents for total RNA isolation --- p.29 / Chapter 2.1.4 --- Reagents for reverse transcription polymerase chain reaction (RT-PCR) --- p.30 / Chapter 2.1.5 --- Reagents and buffers for Western blot analysis --- p.31 / Chapter 2.1.6 --- Vectors for cloning --- p.39 / Chapter 2.1.7 --- Reagents for polymerase chain reaction (PCR) --- p.39 / Chapter 2.1.8 --- Restriction digestion reagents --- p.42 / Chapter 2.1.9 --- Reagents for agarose gel electrophoresis --- p.42 / Chapter 2.1.10 --- Ligation reagents --- p.44 / Chapter 2.1.11 --- Bacterial culture medium --- p.44 / Chapter 2.1.12 --- Dyes and reagents for fluorescent microscope --- p.46 / Chapter 2.1.13 --- Reagents for flow cytometry --- p.48 / Chapter 2.1.14 --- Detection of apoptosis --- p.48 / Chapter 2.2 --- Methods --- p.50 / Chapter 2.2.1 --- Identification of gene expression of candidate genes in HCC --- p.50 / Chapter 2.2.1.1 --- cDNA preparation --- p.50 / Chapter (1) --- Cell culture of HepG2 and WRL-68 cell lines --- p.50 / Chapter (2) --- Total RNA isolation --- p.50 / Chapter (3) --- First-strand cDNA synthesis --- p.51 / Chapter 2.2.1.2 --- RT-PCR of candidate genes --- p.52 / Chapter 2.2.1.3 --- Western blotting --- p.53 / Chapter (1) --- Cell culture --- p.53 / Chapter (2) --- Protein extraction --- p.53 / Chapter (3) --- Quantification of proteins --- p.53 / Chapter (4) --- Detection of RhoC and Rabl4 protein by western blot analysis --- p.54 / Chapter (5) --- Western blotting luminol detection --- p.56 / Chapter 2.2.2 --- Cloning protocol --- p.57 / Chapter 2.2.2.1 --- Amplification of RhoC and Rabl4 genes --- p.57 / Chapter 2.2.2.2 --- Purification of PCR product --- p.58 / Chapter 2.2.2.3 --- Restriction enzymes digestion --- p.53 / Chapter 2.2.2.4 --- Insert/vector ligation --- p.59 / Chapter 2.2.2.5 --- Preparation of chemically competent bacterial cells (E. coli strain DH5a) --- p.60 / Chapter 2.2.2.6 --- Transformation of ligation product into chemically competent bacterial cells --- p.61 / Chapter 2.2.2.7 --- Small-scale preparation of bacterial plasmid DNA --- p.61 / Chapter 2.2.2.8 --- Screening for recombinant clones --- p.62 / Chapter 2.2.2.9 --- DNA sequencing of cloned plasmid DNA --- p.63 / Chapter 2.2.2.10 --- Midi-scale preparation of recombinant plasmid DNA --- p.64 / Chapter 2.2.3 --- Visualization of the subcellular localization patterns --- p.66 / Chapter 2.2.3.1 --- Cell culture of AML12 and HepG2 cell lines --- p.66 / Chapter 2.2.3.2 --- Transfection of GFP fusion constructs into cells --- p.66 / Chapter 2.2.3.3 --- DAPI staining --- p.67 / Chapter 2.2.3.4 --- ER-Tracker´ёØ Blue-White DPX staining --- p.68 / Chapter 2.2.3.5 --- Subcellular localization study using Epi-fluorescence microscopy --- p.68 / Chapter 2.2.4 --- Analysis of cell cycle --- p.69 / Chapter 2.2.4.1 --- Transfection of GFP vectors / GFP-tagged proteins into cells --- p.69 / Chapter 2.2.4.2 --- Analysis of cell cycle by flow cytometry --- p.69 / Chapter 2.2.5 --- Detection of apoptosis --- p.70 / Chapter 2.2.5.1 --- Transfection --- p.70 / Chapter 2.2.5.2 --- Detection of DNA fragmentation --- p.70 / Chapter 2.2.6 --- Reorganization of Actin cytoskeleton by RhoC --- p.71 / Chapter 2.2.6.1 --- Transfection of GFP vectors/GFP-tagged proteins into cells --- p.71 / Chapter 2.2.6.2 --- Rhodamine phalloidin (RP) staining --- p.71 / Chapter 2.2.6.3 --- Epi-fluorescence microscopy --- p.72 / Chapter 2.2.7 --- Analysis of cell invasion under induction of RhoC --- p.72 / Chapter 2.2.7.1 --- "Sub-cloning of human RhoC gene into a mammalian expression vector, pHM6" --- p.72 / Chapter 2.2.7.2 --- Transfection of pHM6-RhoC --- p.73 / Chapter 2.2.7.3 --- Cell invasion assay --- p.73 / Chapter 2.2.8 --- Analysis of downstream effectors in RhoC-mediated pathway --- p.75 / Chapter 2.2.8.1 --- RT-PCR --- p.75 / Chapter 2.2.8.2 --- Western blotting --- p.75 / Chapter 2.2.9 --- Analysis of role of Rabl4 in membrane trafficking --- p.76 / Chapter 2.2.9.1 --- Cloning and transfection --- p.76 / Chapter 2.2.9.2 --- Alexa 594 transferrin conjugate staining --- p.76 / Chapter 2.2.9.3 --- Epi-fluorescence microscopy --- p.77 / Chapter 2.2.10 --- Statistics --- p.77 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Expression of RhoC and Rabl4 in hepatoma cells --- p.78 / Chapter 3.1.1 --- RT-PCR --- p.78 / Chapter 3.1.2 --- Western blotting --- p.81 / Chapter 3.2 --- Subcellular localization of RhoC and Rab 14 --- p.85 / Chapter 3.3 --- Characterization of RhoC --- p.93 / Chapter 3.3.1 --- Cell cycle analysis --- p.93 / Chapter 3.3.2 --- Apoptosis --- p.95 / Chapter 3.3.3 --- Actin cytoskeleton reorganization --- p.97 / Chapter 3.3.4 --- Cell invasion ability --- p.99 / Chapter 3.3.5 --- Downstream effectors of RhoC in cytoskeletal reorganization --- p.102 / Chapter 3.4 --- Characterization of Rabl4 --- p.107 / Chapter 3.4.1 --- Cell cycle analysis --- p.107 / Chapter 3.4.2 --- Apoptosis --- p.109 / Chapter 3.4.3 --- Roles in intracellular transportation --- p.111 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Strong expression of RhoC and Rabl4 in hepatoma cells --- p.117 / Chapter 4.2 --- Subcellular localization of RhoC and Rabl4 --- p.119 / Chapter 4.3 --- The effects of RhoC in normal liver cells --- p.122 / Chapter 4.3.1 --- Cell cycle progression by RhoC through regulating of G1 to S phase transition --- p.122 / Chapter 4.3.2 --- RhoC shows no apoptotic effect in normal liver cell systems --- p.123 / Chapter 4.3.3 --- Formation of actin filaments and stress fibers --- p.124 / Chapter 4.3.4 --- Induction of cell invasion in RhoC-expressing cells --- p.125 / Chapter 4.3.5 --- Downstream effectors in signaling pathway of RhoC in actin filment reorganization and cell invasion --- p.126 / Chapter 4.4 --- The effects of Rabl4 in normal liver cells --- p.132 / Chapter 4.4.1 --- Cell proliferation effects of Rabl4 by increasing percentage of cells in S phase for DNA synthesis --- p.132 / Chapter 4.4.2 --- Rabl4 has no apoptotic effects --- p.133 / Chapter 4.4.3 --- Roles of Rabl4 in vesicular transport --- p.134 / Chapter 4.5 --- Conclusion --- p.138 / Chapter 4.6 --- Future prospects --- p.140 / Appendix --- p.143 / References --- p.147

Yip, Ka Yan. / "August 2011." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 87-113). / Abstracts in English and Chinese. / Acknowledgement --- p.III / Abstract --- p.V / 論文摘要 --- p.VI / Table of Contents --- p.VII / List of Figures --- p.IX / List of Abbreviations --- p.XI / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Hepatocellular carcinoma --- p.1 / Chapter 1.1.1 --- Overview --- p.1 / Chapter 1.1.2 --- Risk factors --- p.3 / Chapter 1.1.3 --- Treatment ofHCC --- p.12 / Chapter 1.2 --- Berberine - a compound derived from Traditional Chinese Medicine --- p.15 / Chapter 1.2.1 --- Traditional Chinese Medicine --- p.15 / Chapter 1.2.2 --- Berberine --- p.16 / Chapter 1.3 --- Cell cycle --- p.18 / Chapter 1.3.1 --- An Overview of cell cycle --- p.18 / Chapter 1.3.2 --- Cell cycle and carcinogenesis --- p.18 / Chapter 1.4 --- Molecular mechanism of apoptosis --- p.20 / Chapter 1.4.1 --- Overview of apoptosis --- p.20 / Chapter 1.4.2 --- Caspases cascade --- p.22 / Chapter 1.4.3 --- Bcl-2 family --- p.24 / Chapter 1.5 --- Apoptosis as a target of cancer therapy --- p.26 / Chapter 1.6 --- Aims of study --- p.27 / Chapter Chapter 2 --- Materials and Methods --- p.28 / Chapter 2.1 --- Cell culture and treatment --- p.28 / Chapter 2.1.1 --- Cell lines --- p.28 / Chapter 2.1.2 --- Berberine --- p.29 / Chapter 2.1.3 --- Chemicals and reagents --- p.29 / Chapter 2.1.4 --- Preparation of solutions --- p.29 / Chapter 2.1.5 --- Procedures --- p.31 / Chapter 2.2 --- Apoptosis detection by FITC Annexin V and PI co-staining --- p.33 / Chapter 2.2.1 --- Chemicals and reagents --- p.33 / Chapter 2.2.2 --- Procedures --- p.33 / Chapter 2.3 --- Gene expression in Berberine-induced apoptotic cells --- p.35 / Chapter 2.3.1 --- Chemicals and Reagents --- p.35 / Chapter 2.3.2 --- Procedures --- p.35 / Chapter 2.4 --- Protein expression in Berberine-induced apoptotic cells --- p.38 / Chapter 2.4.1 --- Chemicals and Reagents --- p.38 / Chapter 2.4.2 --- Preparation of solution --- p.39 / Chapter 2.4.3 --- Procedures --- p.41 / Chapter 2.5 --- Caspase cascade studies in berberine-induced apoptosis --- p.43 / Chapter 2.5.1 --- Chemicals and reagents --- p.43 / Chapter 2.5.2 --- Procedures --- p.43 / Chapter 2.6 --- Cell cycle study in berberine-induced apoptotic cells --- p.44 / Chapter 2.6.1 --- Chemicals and Reagents --- p.44 / Chapter 2.6.2 --- Preparation of solutions --- p.44 / Chapter 2.6.3 --- Procedures --- p.44 / Chapter Chapter 3 --- Results --- p.46 / Chapter 3.1 --- Berberine induces apoptosis in hepatocellular cells --- p.46 / Chapter 3.2 --- Gene expression in Berberine-induced apoptotic cells --- p.53 / Chapter 3.3 --- Caspase cascade studies in berberine-induced apoptosis --- p.58 / Chapter 3.4 --- Protein expression in Berberine-induced apoptotic cells --- p.62 / Chapter 3.5 --- Berberine caused G1 cell cycle arrest in HCC cell lines --- p.65 / Chapter Chapter 4 --- Discussion --- p.76 / References --- p.87

Lee, Ngan Hon. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 120-133). / Abstracts in English and Chinese. / Acknowledgements --- p.IV / Abstract --- p.V / 論文摘要 --- p.VII / Table of Contents --- p.IX / List of Figures --- p.XI / List of Abbreviations --- p.XIII / Chapter 1 Introduction --- p.1 / Chapter 1.1 --- Hepatocellular carcinoma (HCC) --- p.1 / Chapter 1.1.1 --- Risk factors --- p.1 / Chapter 1.1.2 --- Molecular mechanism of HCC --- p.4 / Chapter 1.1.3 --- Treatment of HCC --- p.7 / Chapter 1.2 --- Gambogic acid (GA) - a compound derived from Tradition Chinese Medicine (TCM) --- p.9 / Chapter 1.2.1 --- Traditional Chinese Medicine (TCM) --- p.9 / Chapter 1.2.2 --- Gambogic acid --- p.13 / Chapter 1.3 --- Molecular mechanism of apoptosis --- p.18 / Chapter 1.3.1 --- Overview of apoptosis --- p.18 / Chapter 1.3.2 --- Caspases cascade --- p.18 / Chapter 1.3.3 --- Bcl-2 family --- p.20 / Chapter 1.3.4 --- Mitochondria in apoptosis --- p.23 / Chapter 1.4 --- Apoptosis as a strategy for cancer therapies --- p.26 / Chapter 1.5 --- Aims of study --- p.29 / Chapter Chapter 2 --- Materials and Methods --- p.30 / Chapter 2.1 --- Cell culture and treatment --- p.30 / Chapter 2.1.1 --- Cell lines used --- p.30 / Chapter 2.1.2 --- Gambogic acid (GA) --- p.31 / Chapter 2.1.3 --- Chemicals and reagents --- p.31 / Chapter 2.1.4 --- Preparation of solutions --- p.32 / Chapter 2.1.5 --- Procedures --- p.33 / Chapter 2.2 --- Apoptotic detection --- p.35 / Chapter 2.2.1 --- Chemicals and reagents --- p.35 / Chapter 2.2.2 --- Preparation of solutions --- p.35 / Chapter 2.2.3 --- Procedures --- p.37 / Chapter 2.3 --- Effects of GA on gene expression in HepG2 --- p.41 / Chapter 2.3.1 --- Chemicals and Reagents --- p.41 / Chapter 2.3.2 --- Preparation of solutions --- p.41 / Chapter 2.3.3 --- Procedures --- p.43 / Chapter 2.4 --- Protein expression in GA-induced apoptotic cells --- p.51 / Chapter 2.4.1 --- Chemicals and Reagents --- p.51 / Chapter 2.4.2 --- Preparation of solution --- p.51 / Chapter 2.4.3 --- Procedures --- p.54 / Chapter 2.5 --- Caspase cascade study in GA-induced apoptosis --- p.60 / Chapter 2.5.1 --- Chemicals and reagents --- p.60 / Chapter 2.5.2 --- Procedures --- p.60 / Chapter 2.6 --- Downregulation of mRNA using siRNA vector --- p.62 / Chapter 2.6.1 --- siRNA expression vector --- p.62 / Chapter 2.6.2 --- Chemicals and Reagents --- p.63 / Chapter 2.6.3 --- Preparation of solution --- p.63 / Chapter 2.6.4 --- Procedures --- p.64 / Chapter Chapter 3 --- Results --- p.71 / Chapter 3.1 --- GA induces apoptosis in hepatocellular cells --- p.71 / Chapter 3.2 --- Effects of gene expression in HCC --- p.80 / Chapter 3.3 --- Caspase cascade studies in GA-induced apoptosis --- p.83 / Chapter 3.4 --- Caspase 8 activation in GA-treated cells lead to Bid cleavage --- p.89 / Chapter 3.5 --- GA induces Bax conformational changes and cytochrome c release --- p.95 / Chapter 3.6 --- Levels of protein players involved in apoptosis and cell cycle --- p.101 / Chapter Chapter 4 --- Discussion --- p.106 / References --- p.120

Diao, Shu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 72-73). / Abstracts in English and Chinese. / 摘要 --- p.i / Abstract --- p.ii / Acknowledgements --- p.iv / Contents --- p.viii / Chapter Chapter 1 --- Introduction and Background / Chapter 1.1 --- General introduction to miRNA --- p.1 / Chapter 1.1.1 --- The discovery and biogenesis of miRNA --- p.1 / Chapter 1.1.2 --- The function of miRNA --- p.3 / Chapter 1.2 --- The liver-specific miRNA : miR-122a --- p.5 / Chapter 1.2.1 --- Discovery and biogenesis of miR-122a --- p.5 / Chapter 1.2.2 --- "miR-122a, a liver specific miRNA" --- p.6 / Chapter 1.2.3 --- miR-122a and Hepatocellular carcinoma (HCC) --- p.6 / Chapter 1.2.4 --- Therapeutic opportunities and challenges of miR-122a --- p.7 / Chapter 1.3 --- Techniques and approaches to study miRNAs --- p.8 / Chapter 1.3.1 --- Discovery of novel miRNA --- p.8 / Chapter 1.3.2 --- Detection of miRNA --- p.9 / Chapter 1.3.3 --- Functional study of miRNAs --- p.10 / Chapter 1.3.4 --- Prediction and validation of miRNA targets --- p.10 / Chapter 1.4 --- References --- p.12 / Chapter Chapter 2 --- Materials and methods / Chapter 2.1 --- Cell culture --- p.18 / Chapter 2.2 --- Cell transfection --- p.18 / Chapter 2.3 --- RNA extraction --- p.18 / Chapter 2.4 --- Plasmid extaction --- p.19 / Chapter 2.5 --- Competent cell preparation --- p.19 / Chapter 2.6 --- Bacterial Transformation --- p.20 / Chapter 2.7 --- Purification of DNA fragments from agarose gel --- p.21 / Chapter 2.8 --- Genomic DNA extranction from MIHA cell --- p.21 / Chapter 2.9 --- Real-time RT-PCR analysis --- p.21 / Chapter 2.10 --- Lenti-vector Construction for miRNA expression --- p.22 / Chapter 2.11 --- Lentivirus production --- p.22 / Chapter 2.12 --- Lentiviral vector titering --- p.23 / Chapter 2.13 --- Bradford protein assay --- p.23 / Chapter 2.14 --- Western Blot --- p.24 / Chapter 2.14.1 --- Sample preparation --- p.24 / Chapter 2.14.2 --- Gel electrophoresis --- p.24 / Chapter 2.14.3 --- Blocking --- p.25 / Chapter 2.14.4 --- Incubation with Primary and Secondary Antibodies --- p.25 / Chapter 2.14.5 --- Substrate Incubation --- p.26 / Chapter 2.14.6 --- Exposeto x-ray film --- p.26 / Chapter 2.15 --- MTT Cell Proliferation Assay --- p.26 / Chapter 2.16 --- Apoptosis analysis :DAPI Staining --- p.26 / Chapter 2.17 --- 2-D Protein Gel Electrophoresis and MS --- p.26 / Chapter 2.17.1 --- Materials --- p.27 / Chapter 2.17.2 --- Protein extraction --- p.27 / Chapter 2.17.3 --- 2-D Electrophoresis --- p.27 / Chapter 2.17.4 --- Gel staining and image analysis --- p.28 / Chapter 2.17.5 --- In-gel protein digestion with trypsin --- p.28 / Chapter 2.17.6 --- MALDI-TOF MS and database search --- p.28 / Chapter 2.18 --- Statistical Analysis --- p.29 / Chapter Chapter 3 --- Expression of HCC-associated miRNA in HCC cell lines / Chapter 3.1 --- Introduction --- p.30 / Chapter 3.2 --- Experimental Section --- p.31 / Chapter 3.2.1 --- Cell culture --- p.31 / Chapter 3.2.2 --- RNA extraction --- p.31 / Chapter 3.2.3 --- miRNA-specific quantitative Real-time PCR --- p.31 / Chapter 3.3 --- Results and Discussion --- p.32 / Chapter 3.3.1 --- Expression of miR-let7a in HCC cells --- p.32 / Chapter 3.3.2 --- Expression of miR-221 in HCC cells --- p.33 / Chapter 3.3.3 --- "Expression of miR-122a,a liver-specific miRNA in HCC cells" --- p.34 / Chapter 3.4 --- Conclusions --- p.35 / Chapter 3.5 --- References --- p.36 / Chapter Chapter 4 --- Ectopic overexpression of miR-122a in HCC cells / Chapter 4.1 --- Introduction --- p.38 / Chapter 4.2 --- Experimental Section --- p.38 / Chapter 4.2.1 --- Overexpression of miR-122a with mimics --- p.38 / Chapter 4.2.2 --- Lentivirus-mediated miR-122a expression --- p.40 / Chapter 4.2.3 --- RNA extraction --- p.44 / Chapter 4.2.4 --- Expression level of miR-122a after transduction --- p.44 / Chapter 4.2.5 --- Western Blot --- p.44 / Chapter 4.3 --- Result and discussion --- p.46 / Chapter 4.3.1 --- Expression level of miR-122a after transfection --- p.46 / Chapter 4.3.2 --- Lentivirus-mediated miR-122a expression --- p.47 / Chapter 4.4 --- Conclusions --- p.52 / Chapter 4.5 --- References --- p.54 / Chapter Chapter 5 --- Gain-of-function study of miR-122a in HCC cells / Chapter 5.1 --- Introduction --- p.55 / Chapter 5.2 --- Experimental Section --- p.56 / Chapter 5.2.1 --- Cell culture --- p.56 / Chapter 5.2.2 --- Cell transfection --- p.56 / Chapter 5.2.3 --- Lentiviral vector transduction --- p.56 / Chapter 5.2.4 --- MTT Cell Proliferation Assay --- p.56 / Chapter 5.2.5 --- Apoptosis analysis - DAPI Staining --- p.57 / Chapter 5.2.6 --- 2-D Protein Gel Electrophoresis and MS --- p.57 / Chapter 5.2.7 --- miRNA target prediction using bioinformatic approaches --- p.59 / Chapter 5.3 --- Result and discussion --- p.59 / Chapter 5.3.1 --- Phenotypic changes of HepG2 cells caused by ectopic overexpression of miR-122a --- p.59 / Chapter 5.3.2 --- miR-122a target prediction using bioinformatic approaches --- p.62 / Chapter 5.3.3 --- Experimental validation of miR-122a targets by proteomics approach --- p.67 / Chapter 5.4 --- Conclusion --- p.70 / Chapter 5.5 --- References --- p.71 / Appendix --- p.73

在世界範圍內，肝細胞癌(HCC)是其中一種惡性程度很高並且預後很差的疾病。和其他的癌症一樣，肝癌是一种基因疾病，基因異常的積累在肝癌的生成中扮演著重要的角色。近年來，第二代測序技術（NGS）的迅速發展顯現了前所未見的能力揭示癌癥基因組中分子的複雜性，這為癌癥的生物學，診斷和药物治療提供了一個嶄新的思路。 / 非酒精脂肪性肝炎（NASH）是一種與代謝有關的疾病，在發達國家地區例如美國，歐洲，日本和加拿大，這是其中一種越來越常見的HCC 的病因。在本論文的第一部份，三個NASH 相關的HCC 以及其配對的血DNA 進行了全基因組測序（WGS）。另外還有一個來源于這三個NASH 相關的HCC其中之一的細胞株也進行了全基因組測序。在全部樣品中，測序深度範圍介乎29.1X 到102.2X，序列覆蓋度均大於99%。結果顯示發現的新的單核苷酸變異（SNVs）數量介乎于6,898 至17,129，平均值是11,133。根據這些找到的SNV，隨機選出56 個體細胞SNV 進行Sanger 測序，其中92.9%可以被確認。基因突變譜顯示有頻繁的A:T>G:C 和C：G>A:T 體細胞突變，而C：G>T:A 則在CpG 位點頻繁出現。在眾多的非同義體細胞突變基因中，我們選擇了CTNNB1，PNLIP 和MLL2 這三個基因進行進一步研究，此三個基因都在多於一個病例中發現有突變。在額外的一組44 對NASH 相關HCC及50 對HBV 相關的HCC 的癌變組織和其臨近非腫瘤組織中，我們進一步對這三個基因和TP53 的編碼區域進行了測序，而TP53 是在HBV 相關HCC中被報導有高頻率突變的。在NASH 相關的HCC 中，這些基因都只在腫瘤組織中發現重複出現的錯義突變，在臨近的非腫瘤肝組織沒有發現有突變。在NASH 相關的HCC 中，CTNNB1 的突變率（36.4%）明顯高於在HBV相關的HCC 中的突變率（12.0%，P=0.007）。PNLIP 和MLL2 的突變只在NASH相關的HCC 中發現，其突變率分別為12.1%和7.1%，而在HBV 相關的HCC中，則沒有發現突變。然而，TP53 的突變率在NASH 相關的HCC 及HBV相關的HCC 中差別不明顯（P>0.1）。在功能性研究的實驗中，我们发现在HKCI-10（有PNLIP 突變D396N）細胞株中，PNILP 的活性比在正常肝細胞L02 細胞株（野生型PNLIP）中要低。在永生性肝細胞L02 細胞株中，CTNNB1的突變引起了TOPFLASH 活性的提高以及增加了細胞群落形成的能力。HKCI-10 是一條我們實驗室建立的NASH 相關的肝細胞癌細胞株，在HKCI-10細胞株中，抑制CTNNB1 表達引起了細胞生長和增殖的減少。另外，在DEN誘導肝癌的有代謝失衡的db/db 轉基因鼠中，發現了一個CTNNB1 的突變T41A。據報導，CTNNB1 發生的致癌突變會引起蛋白的穩定並且因此激活經典的Wnt/β-catenin 信號通路，從而引致特定基因的轉錄。對於CTNNB1中最常見的突變S45P(在發現的CTNNB1 突變中占31.3%)，我們做了ChIP-array 實驗，結果顯示，在HKCI-10（CTNNB1 有S45P 突變）中，CTNNB1比在Hep3B 中（野生型CTNNB1）有著更緊密的啟動子結合能力（P<0.001）。Gene ontology 分析結果表明，被S45P 富集的生物過程涉及有RNA 代謝調節，轉錄因子活性和凋亡。MYC，E2F1 和ZFX 被ChIP-PCR 證實是與CTNNB1突變子S45P 有著更緊密結合能力的轉錄因子。 / 在本論文的第二部份，爲了進一步闡明致癌性的CTNNB1 突變S45P在HCC 中的角色，我們研究了一個此前未在HCC 報導過的基因ZFX。ZFX是一個在脊椎動物中高度保守，屬於Zfy 家族的zinc finger 蛋白。有報導指出ZFX 對於胚胎和造血幹細胞的自我更新有著重要的作用。另外亦有文獻報導ZFX 在一系列人類癌癥病例中有過度表達，例如食道癌，胃癌，前列腺癌和神經膠質瘤，並且顯現出致癌基因的特性。在本研究中，qRT-PCR結果提示ZFX 在HCC 腫瘤中的表達明顯高於正常肝組織。ZFX 的表達在51.8%的HCC 腫瘤中明顯高於其配對的鄰近非腫瘤肝組織。功能性研究表明，在MTT 實驗中，細胞的生存力在ZFX 缺失的穩定克隆中明顯減弱（P<0.0001）。在細胞群落形成實驗中，ZFX 缺失的穩定克隆顯示出明顯減弱的群落生長能力（P<0.0001）。在單細胞克隆生成實驗中，ZFX 缺失的HCC穩定克隆展示出數量更少，體積更小的細胞群落。另外，ZFX 基因抑制或者cisplatin 單獨處理均顯示細胞生存力的抑制，其抑制效率分別是24.0%和30.9%。當ZFX 基因抑制合併cisplatin 處理時，細胞生存力的抑制效率顯著地提高至65.2%，這個結果提示ZFX 基因抑制和cisplatin 處理兩者有協同增效作用（P<0.0001）。ZFX 基因的缺失會引起兩個為人熟知的胚胎幹細胞（ESCs）標誌物SOX-2 和NANOG 表達的明顯降低。 / 綜上所述，通過進行全基因測序，本論文的研究結果為NASH 相關的HCC 分子層面上的異常提供了一個高解析度的視覺角度。在NASH 相關的HCC 中，一些可能對於肝細胞癌變有重要作用並且重複出現突變的基因被確定，例如CTNNB1 和PNLIP。CTNNB1 的突變體S45P 的其中一個下游目標基因ZFX，在HCC 中被證實有幹細胞和腫瘤啟動細胞特性。闡明在HCC發展過程中的分子改變以及機制，對於為肝癌病人探索新的治療手段有著重要意義。 / Hepatocellular Carcinoma (HCC) is one of the most malignant diseases worldwide with poor prognosis. Like other human cancers, HCC is a genetic disease, where accumulation of genetic aberration plays important role in the liver carcinogenesis. The rapid advances in Next Generation Sequencing (NGS) technology in recent years have allowed unprecedented ability to unravel the molecular complexity of the cancer genome, providing new insights into the cancer biology, diagnosis and therapeutic drug development. / Non-alcoholic steatohepatitis (NASH), which is related to metabolic disorder, is an increasing common etiological factor of HCC, especially in developed countries such as United States, Europe, Japan and Canada. In first part of this thesis, Whole Genome Sequencing (WGS) was performed on three NASH-associated HCCs and their matched lymphocytic DNA. A cell line developed from one of the three NASH-associated HCCs was also subjected to WGS. The sequencing depth ranged from 29.1X to 102.2X with the coverage >99% shown in all samples. Novel SNVs identified ranged from 6,898 to 17,129 with an average of 11,133. Based on the SNVs found, the validation rate was 92.9% in 56 randomly selected somatic SNVs by Sanger sequencing. Mutational spectrum showed frequent somatic substitution of A:T>G:C and C:G>A:T while C:G>T:A transition exhibited a predominant somatic mutation rate in CpG sites. Among the non-synonymous somatic mutated genes, we selected CTNNB1, PNLIP and MLL2 which were mutated in more than one tumor for further study. In additional cohort of 44 NASH-associated and 50 HBV-associated HCC tumors and adjacent non-tumoral tissues, further sequencing all the coding regions of these three genes and TP53, which has been reported to be highly mutated in HBV-associated HCCs, were carried out. In NASH-associated HCCs, all genes harboured recurrent missense mutations exclusive in tumor but none in adjacent ,non-tumoral liver. The prevalence of CTNNB1 mutations was significantly higher in NASH-associated HCCs (36.4%) when compared to HBV-associated HCCs (12.0%, P=0.007). Mutations of PNLIP and MLL2 were detected only in NASH-associated HCCs with rates of 12.1% and 7.1%, respectively, but none in HBV-associated HCCs. The mutation rate of TP53, however, did not differ much between NASH-associated and HBV-associated HCCs (P>0.1). In functional study, for PNLIP, a loss-of-function in PNLIP activity was found in HKCI-10 harbouring D396N mutation as compared to normal liver cell, L02 with wild-type PNLIP. We demonstrated that CTNNB1 mutants conferred elevated TOPFLASH activity and enhanced colony growth in an immortalized hepatocyte cell line L02. Knockdown of CTNNB1 in HKCI-10, which is a NASH-associated HCC in-house cell line, resulted in inhibition of cell growth and proliferation. Also, a CTNNB1 mutation (T41A) was found in DEN-induced liver cancer of db/db transgenic mouse with metabolic disorder. Since oncogenic mutations of CTNNB1 were reported to be contributed to the stabilization of the protein and hence activate the canonical Wnt/β-catenin signaling pathway, inducing transcription of specific gene sets. We performed ChIP-array focusing on the most common CTNNB1 S45P mutation (accounted for 31.3% of all detected CTNNB1 mutants) in NASH-associated HCCs, the result showed more intense promoter binding affinities in HKCI-10 with CTNNB1 S45P mutation than Hep3B with wild-type CTNNB1 (P<0.001). Gene ontology analysis revealed that S45P mutant enriched the process including RNA metabolic regulation, transcription factor activities and apoptosis. Furthermore, we found that CTNNB1 S45P mutant showed more profound binding affinity to the promoter regions of transcription factors MYC, E2F1 and ZFX by ChIP-PCR. / In the second part of the thesis, we aimed to further understand the role of oncogenic CTNNB1 S45P mutant in HCC. Zinc finger protein X-linked (ZFX) gene which is previously undescribed in HCC was studied. ZFX is a zinc finger protein of Zfy family that is highly conserved among vertebrates. It has been reported that ZFX is required for self-renewal of embryonic and hematopoietic stem cells. Also, ZFX is suggested to be overexpressed in a number of human cancers such as esophageal carcinoma, gastric cancer, prostate cancer and glioma, conferring oncogenic characteristics. In this study, qRT-PCR analysis showed a significant higher ZFX expression in HCC tumors compared to normal livers. 51.8% of HCC tumors showed significant up-regulations of ZFX when compared to paired adjacent non-tumoral livers. Functional studies showed significant reduction in in-vitro cell proliferation in HCC by MTT assay (P<0.0001) and colony formation ability by colony formation assay (P<0.0001) in ZFX-deficient stable clones. In single-cell clonogenic assay, ZFX-depleted HCC exhibited fewer and smaller colonies. In addition, while ZFX knockdown or cisplatin treatment alone showed an inhibitory effect on cell viability by 24.0% and 30.9%, respectively, the reduction efficiency of cell viability increased dramatically to 65.2% when combine ZFX-knockdown and cisplatin treatment, indicating a synergistic effect between them (P<0.0001). Also, significant reduced expressions of SOX-2 and NANOG, both well-known embryonic stem cells (ESCs) markers, were observed as a result of ZFX depletion. / In summary, findings from this thesis provided high-resolution insight into the molecular aberrations in NASH-associated HCCs by performing WGS. Recurrent mutated genes which may be of great importance in hepatocellular carcinogenesis induced by NASH were identified, such as CTNNB1 and PNLIP. One of the S45P CTNNB1 downstream targets ZFX was demonstrated to confer stemness and tumor-initiating cells (TICs) characteristics in HCC. The understanding of molecular changes and mechanisms underlying HCC development will thus facilitate the exploration of new therapeutic options in patients with this deadly disease. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Jiawei. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 166-190). / Abstracts also in Chinese.

SND1 MEDIATED DOWNREGULATION OF PTPN23 IN HEPATOCELLULAR CARCINOMA By Nidhi Jariwala, MS A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University, 2014. ADVISOR: Dr. Devanand Sarkar Associate Professor, Department of Human and Molecular Genetics Blick Scholar Associate Scientific Director, Cancer Therapeutics VCU Institute of Molecular Medicine Massey Cancer Center ABSTRACT Staphyloccocal nuclease domain containing protein 1 (SND1) is identified as an oncogene in multiple cancers, including hepatocellular carcinoma (HCC). SND1 regulates gene expression at transcriptional as well as post-transcriptional level and mediates molecular pathways that culminate into carcinogenesis. SND1 is a component of RNA-induced silencing complex (RISC) and functions as a nuclease for RNAi-mediated mRNA degradation. On the other hand SND1 also binds to specific mRNAs, increasing their stability and hence expression. The aim of the present study is to identify mRNAs to which SND1 binds and modulates them either by degradation or increasing stability which might facilitate promotion of HCC by SND1. We performed RNA immunoprecipitation followed by RNA sequencing (RIP-Seq) using anti-SND1 antibody and human HCC cell line QGY-7703. More than 350 mRNAs were identified to be interacting with SND1, of which Protein tyrosine phosphatase non-receptor 23 (PTPN23) was of particular interest, since PTPN23 has been identified to be a tumor suppressor and its role in HCC has not been studied. We document that SND1 can bind to PTPN23 mRNA and induce its degradation. There is an inverse correlation between SND1 and PTPN23 levels in human HCC cell lines and PTPN23 level is downregulated in HCC. Our study thus identifies a novel mechanism by which SND1 promotes hepatocarcinogenesis and identifies PTPN23 as a potential tumor suppressor in HCC. Further studies need to be performed to explore the relationship of these two molecules in in vivo models and to develop PTPN23 overexpression as a potential therapeutic approach for HCC.

acase@tulane.edu / Introduction: High-Intensity Focused Ultrasound (HIFU) is the only noninvasive method available today for thermal ablation of tumors. HIFU-induced rapid heating and mechanical disruption of tissue, not only has a direct destructive effect on tumors, but also provides a noninvasive way for targeted release of chemotherapeutic drugs from drug delivery vehicles such as temperature sensitive liposomes (SfTSLs). The objective of this work was to evaluate the synergistic treatment of Sorafenib-loaded TSLs (SfTSLs) and HIFU via in vitro analysis of cell viability and proliferation using an aggressive human liver cancer cell line and corresponding in vivo analysis of tumor growth and survival using a human xenograft mouse model. Materials and Methods: Liposomes were developed using 70% Dipalmitoylphosphatidylcholine, 20% L-a-Phosphatidylcholinehydrogenated Soy, and 10% Cholesterol using thin film hydration method to encapsulate Sorafenib at 10μM. Pellets of Hep3B human liver cancer cells (100 μl, 2.7 million cells/ml) were placed in a 0.2 ml thin-wall PCR tube to mimic dense tumor aggregation. Cell pellets were then inoculated with HIFU alone, SfTSLs, or exposed to a combination of HIFU and SfTSLs. The focused ultrasound signal was generated by a 1.1 MHz transducer with acoustic power ranging from 4.1 W to 12.0 W. Cell viability and proliferation experiments were conducted to measure cancer cell damage at 24, 48, 72, and 96 h post treatment via Annexin V/PI and WST-8 staining. In our in vivo study, 1.0×106 Hep3B cells in Matrigel were injected into left and right flanks of athymic nude mice. Tumors were allowed to grow to 8-10 mm size and then separated into the following treatment groups: HIFU alone, SfTSLs (50 μl) alone, SfTSLs + HIFU, and sham. Tumor sizes were measured by caliper every day and a diagnostic ultrasound system was used pre-treatment, 5 days, 14 days, and prior to sacrificing. Tumors were grouped and processed at 5 days, 14 days, or placed in a survival study to evaluate whether treatment facilitated longer lifespans. Tumor tissues were collected for H&E staining and evaluated by a blinded pathologist post euthanasia. Results and Discussion: Our in vitro data indicate that Hep3B cells exposed to both SfTSLs and HIFU have a significantly lower viability and proliferation rate than untreated cells or the cells treated with only SfTSLs or HIFU. According to our in vivo study, tumor growth in the SfTSLs + HIFU group was reduced as compared to Sham, SfTSLs only, or HIFU only groups. Conclusions: The results of our in vitro and in vivo experiments clearly indicate that chemotherapeutic drug-loaded SfTSLs and HIFU can be an effective therapy for locally aggressive liver cancer. This combination treatment leads to more cellular damage, reduction in tumor growth, and better survival. / 1 / Gray Halliburton

Both primary and secondary liver cancers are common and the majority of patients are not eligible for surgical resection or a liver transplant, which are considered the only hope of cure. Mortality rates are high and there is a need for alternative treatment options. New forms of local treatment work best on small tumours; large ones, however, remain difficult to treat. Hyperthermia involves heating tumours to 40??-44?? C. The aim is to heat the entire tumour without damaging the surrounding normal tissue. Treating deep seated tumours is technically challenging. Ferromagnetic embolisation hyperthermia (FEH) is a novel method of treating liver tumours. Magnetic microspheres are infused into the hepatic artery and lodge primarily in the tumour periphery. An applied alternating-current magnetic field causes the microspheres to heat. Animal experiments have shown that this is a promising technique. There is a need for modelling of FEH prior to commencement of clinical trials. Analytical and numerical models of tumour heating during FEH treatment are presented here. The models help predict the temperature distributions that are likely to arise during treatment and give insight into the factors affecting tumour and liver heating. The models incorporate temperature-dependent thermal properties and blood perfusion rates of the tissues and a heterogeneous clustering of microspheres in the tumour periphery. Simulations show that the poorly perfused tumours heat preferentially while the liver is effectively cooled by blood flow from the portal vein. A peripheral distribution of heat sources produces a more even temperature field throughout the tumour, compared to a heat source that is centred within the tumour core. Large tumours reach higher temperatures and have higher heating rates, supporting experimental findings. Using temperature-dependent, rather than constant, values for thermal conductivities and blood perfusion rates results in higher temperatures within the tumour. The uneven clustering of microspheres in the tumour periphery leads to a more heterogeneous temperature distribution in the core, but it has less of an effect on the wellperfused liver. The results show that FEH has the potential to effectively treat liver tumours and the technique merits further investigation.

No description available.

At the beginning of the 21st century, primary liver cancer (PLC) remains the fifth most common malignancy in men worldwide, and the eighth in women. Central Africa and South East of Asia are high risk geographic areas for PLC, whereas developed countries appear to be generally low risk. Infections with hepatitis B (HBV) and C (HCV) viruses are the main risk factors for PLC, accounting for well over 80% of PLC cases detected worldwide. The recently detected increase in both incidence and mortality by PLC in developed countries is strongly related to these viral infections. The evaluation of PLC time trends needs to take into consideration the geographic distribution and effect of these viruses. This thesis presents three studies which the aim to describe PLC incidence and mortality issues in different geographic areas, each addressing several epidemiological and methodological issues. For each study, different statistical methods on the basis of the Bayesian inference have been proposed, evaluated and discussed in order to cope with extra-Poisson variability. The first study, entitled "Meta-analysis of cohort studies of risk of liver cancer death among HBV carriers", evaluates the variability in PLC mortality reported in 11 cohort studies of male HBV carriers, taking into consideration the effects of geographic area and the choice of the general population versus a more comparable group such as HBV-negative workers or blood donors as the comparison group. The statistical methods of this study focuses on mixtures of Poisson distributions. The "stickbreaking" method has been used to estimate the number of components of the mixture of Poisson distributions, and, thus to obtain a pooled relative risk (RR) of death for PLC among male HBV carriers. The pooled RR of death by PLC related to HBV infection was 23.5 (95% Credibility Interval (CRI): 14.9 - 44.5). Studies carried out in high risk areas for PLC (China and Taiwan) showed RRs 2 to 5-fold higher than those of studies carried out in Europe, Japan and the U.S.. In low risk areas for PLC, studies which used workers or blood donors as comparison groups had RRs 1.9-fold higher (95% CRI: 1.2 - 3.1) than studies which used the general population. However, in high risk areas, the ratio of RRs was 5.3-fold (95% CRI: 3.4 - 7.9). This is the first time that a "healthy donor effect" has been quantified in longitudinal studies. The second study, entitled "Geographic distribution of primary liver cancer in Europe in 2002" evaluates the effect of HBV and HCV seroprevalence in 38 European countries on PLC incidence and mortality. Mixed Poisson models based on Bayesian inference have been used to smooth Standardized Incidence (SIR) and Mortality (SMR) ratios for PLC accounting for the effect of HBV and HCV prevalences. This approach enabled us to both examine the effect of different levels of HBV and HCV, and to identify remaining variability in PLC after accounting for infection rates. Bayesian inference allowed the determination of posterior probabilities for the somoothed SIRs and SMRs (hereafter RRs). The Deviance Information Criterion (DIC) and the "effective number of parameters" (pD) have been used as tools for model choice. The highest mortality and incidence PLC RRs were found in Southern European countries (RR range 0.9-2.4), whereas Northern European countries showed the lowest RRs (RR range: 0.3-0.9). The effect of HBV infection was not found to be statistically significant in the model which accounted for both HBV and HCV prevalence. Countries with a prevalence of HCV higher than 2% (e.g.: Italy and Spain) had a higher risk of incidence and mortality (RR range: 1.28 - 1.78) than countries with HCV prevalence below 1%. Thus, the high risk of PLC detected in Southern Europe appears to be explained, in part, by HCV infection. The high HCV seroprevalence in this area could be associated with exposure 30-50 years ago. There may be an underestimation of PLC incidence and mortality rates in Eastern European countries given the low PLC RRs reported, despite high HBV and HCV seroprevalences observed. The implementation of population-based cancer registries in Eastern European countries is warranted, as well as HCV prevalence studies across Europe, to better determine the distribution of PLC in Europe and its relationship with that virus. The last study, entitled "Time trends in liver disease in Spain during the period 198397", describes incidence and mortality trends in hepatocellular carcinoma and cholangiocarcinoma as well as mortality trends in liver cirrhosis in Spain. Autoregressive age-period-cohort (APC) models have been used to evaluate the time trends. We found that APC models performed well for those liver diseases with large number of cases, whereas the age-period models did for those liver diseases with low number of cases. We found an increase in incidence and mortality of hepatocellular carcinoma in Spain (annual percent change (APCH) in men's incidence: 6.6%, 95% CRI: 5.8, 8.1: APCH in women's incidence: 4.5%, 95% CRI: 1.4%, 7.3%; APCH in men's mortality: 6.8%, 95% CRI: 5.8%, 8.1%; APCH in women's mortality: 5.1%, 95% CRI: 3.5%, 6.3%), that appear to be related to HCV exposure 30 years ago, as described in other studies of PLC. We also found an increasing trend in cholangiocarcinoma mortality (APCH in men: 17.1%, 95% CRI: 13.5%, 21.2%; APCH in women: 15.0%, 95% CRI: 11.5%, 19.5%) similar to that found in some developed countries, that could be attributed to improvement in diagnosis resulting from better imaging and diagnostic techniques. However, we did not detect a significant increasing trend in cholangiocarcinoma incidence, perhaps due to the low number of cases reported by the Spanish cancer registries. We have observed a decreasing trend in cirrhosis mortality in both sexes during the study period (APCH in men: -3.1%, 95% CRI: -5.1, -1.9%; APCH in women: -2.9%; 95% CRI: -6.2%, -1.3%), although younger cohorts did not show this pattern. This cohort effect suggests the possibility that younger cohorts could be exposed to some additional risk factors besides alcohol consumption. HIV and HCV or HBV co-infection and intravenous drug addiction could explain the increase in liver cirrhosis mortality among younger cohorts. The flexibility of the Bayesian approach allowed us to cope with extra-Poisson variability in three statistical analyses, applying different models, and addressing relevant methodological aspects specific to each problem. Challenging statistical issues in the framework of Bayesian applied modelling are: i) the selection of prior distributions for model parameters, which is related to convergence of the model; and ii) model selection procedures, and these remain important considerations for future research.