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Hnf4α and Choline Metabolism Role in β-catenin Activated Liver Carcinogenesis / Le rôle d’Hnfα et du métabolisme de la choline dans les carcinomes hépatocellulaires activés pas la β-caténineSartor, Chiara 24 September 2015 (has links)
La voie de signalisation WNT/β-caténine est impliquée dans de nombreuses processi cellulaires, du développement à la physiologie. Dans le foie adulte, elle est nécessaire pour établir et maintenir la zonation métabolique, condition préalable pour la fonctionnalité de l’organe, mais elle est aussi cause d’un pourcentage non négligeable (11-32%) de carcinomes hépatocellulaires (CHC), qui surviennent après mutation activatrice du gène codant la β-caténine. Mes travaux se sont inscrits dans la continuité de travaux de l’équipe auxquels j’ai participé , et ont eu pour principaux objectifs : (1) d’explorer le rôle du facteur de transcription Hnf4α dans la physiologie et les cancers du foie, en lien avec la signalisation β-caténine ; (2) de déterminer si une imagerie tumorale par tomographie par émission de positrons (TEP) spécifique de la captation de choline pouvait prédire les CHC mutés pour la β-caténine et si le métabolisme de la choline pouvait présenter une piste thérapeutique des cancers du foie.Pour ces deux projets, j’ai eu accès à des cohortes de patients atteints de cancers du foie, mais j’ai également pu bénéficier du modèle gain-de-fonction de la β-caténine développé au laboratoire, qui consiste en une perte du suppresseur de tumeur Apc, frein majeur de la voie β-caténine conduisant à des cancers du foie. Grâce à un modèle d’invalidation hepato-spécifique et conditionnelle du gène Hnf4α, j’ai pu prouver que la perte de Hnf4α mène à une augmentation de la prolifération, du stockage des lipides dans le foie et à une désorganisation de l’architecture zonale hépatique, en particulier celle de la triade portale. J’ai aussi démontré que dans un contexte de carcinome murin invalidé par Apc, le rôle suppresseur de tumeur d’Hnf4α était mineur.Une approche métabolomique avait montré qu’un signal β-caténine perturbait le métabolisme des phospholipides dérivant de la choline. Grâce à une étude parallèle réalisée chez des patients porteurs de CHC d’une part, et dans nos modèles murins d’autre part, nous avons pu mettre en évidence par TEP une fixation accrue de la F-choline dans les tumeurs activées β-caténine. Ce phénotype est spécifique d’une signalisation β-caténine active puisque cette captation accrue n’était pas présente chez les patients porteurs de carcinome hépatocellulaire non mutés ou chez les souris présentant une cancérogenèse indépendante de la β-caténine (modèle DEN). J’ai ensuite étudié le devenir intracellulaire de la choline. En utilisant de la choline radiomarquée j’ai montré in vitro qu’un signal β-caténine aberrant accroit l’incorporation de choline dans les phospholipides, et accroit également son rôle de donneur de groupements méthyles, participant à la méthylation de l’ADN. Cela pourrait expliquer pourquoi l’ADN est hyperméthylé chez les souris avec la perte d’Apc, puisque l’administration d’un régime sans choline et methionine à ces souris réverse le phénotype d’hyperméthylation. L’ensemble de ces résultats suggère que la choline pourrait jouer un rôle important dans la cancérogenèse liée à la β-caténine. Nous proposons que des TEP F-choline pourraient être utilisés pour diagnostiquer les CHC mutés β-caténine, et à terme des thérapies ciblées sur ce métabolisme pourraient être envisagées. / WNT/β-catenin is a pillar during development and in adult physiology. In particular in the adult liver it is a double-edged sword: it is necessary to establish the metabolic zonation, requirement for having a functional organ, but it is also involved in the onset of 11-32% of hepatocellular carcinoma (HCC). My thesis work has been based on the team previous results and it is focused on two main subjects: (1) the first aim was to decipher the role of Hnf4α both in physiology and in HCC development and its relationship with WNT/β-catenin signalling and (2) the second part explores the possible use of Fluoro-choline (FCh) positron emission tomography (PET) in the diagnosis of β-catenin-activated liver tumours.In this study I used cohorts of patients having HCC, but also inducible and hepatospecific knock-out mice for adenomatous polyposis coli (APC) gene (thereafter called ApcKO mice). Apc is the most important negative regulator of β-catenin, and it hepatic loss leads to aberrant activation of β-catenin, disrupting liver zonation and initiating long-term liver cancers. I generated also inducible hepatospecific Hnf4α knock-out mice and I demonstrated an increased proliferation, lipids accumulation and disorganization in the portal triad architecture, together with a mild distruption of liver zonation. Then, looking at cancer onset, I demonstrated that Hnf4α loss is not able per se to initiate liver cancer, and has no tumour suppressor role in β-catenin activated tumours onset and progression.We performed a metabolic analysis of ApcKO livers, showing that β-catenin is able to deregulate lipids metabolism, in particular that of phospholipids derived from choline. In collaboration with clinicians, I studied human patients who underwent FCh/PET, showing that β-catenin-mutated tumours had an increased uptaken of F-Choline whereas non-mutated β-catenin human HCC had not. Similar results were obtained with mice, either ApcKO β-catenin-activated HCC or β-catenin-independent mice HCC, obtained through a N-diethylinitrosamine (DEN) injection.Choline in cells splits in two main pathways: it is both a methyl-group donor and a precursor for phospholipids production. I tested this through radiolabeled fluxes in in vitro experiments. In β-catenin activated hepatocytes and tumours there are more phospholipids and more methyl groups in DNA derived from choline than in control mice. Moreover in ApcKO DNA is hypermethylated, and it is dependent on choline supply from diet.All these results together show the importance for β-catenin activated tumours to have a supply in choline, and so open a way not only in PET exploitation for having a precise diagnosis, but also in deciphering the importance of choline pathway, to possibly develop a targeted therapy.
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Delineation of genomic imbalances on chromosome 1 and 4q in hepatocellular carcinoma.January 2003 (has links)
Leung Ho-yin. / Thesis submitted in: July 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 104-118). / Abstracts in English and Chinese. / Acknowlegements --- p.i / Abstract (English) --- p.ii / Abstract (Chinese) --- p.iv / "Table of Contents," --- p.vi / List of Figures --- p.xi / List of Tables --- p.xii / Abbreviation --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 . --- Cancer Incidences in Hong Kong --- p.2 / Chapter 1.2. --- Hepatocellular Carcinoma (HCC) --- p.2 / Chapter 1.3. --- "Etiological Risk Factors," --- p.7 / Chapter 1.3.1. --- Liver Cirrhosis / Chapter 1.3.2. --- Chronic Viral Hepatitis / Chapter 1.3.2.1. --- Hepatitis B Virus (HBV) / Chapter 1.3.2.2. --- Hepatitis C Virus (HCV) / Chapter 1.3.3. --- Dietary Aflatoxin B1 exposure / Chapter 1.3.4. --- Heavy Alcohol Consumption / Chapter 1.3.5. --- Hemochromatosis / Chapter 1.4. --- Genetic Aberration in HCC --- p.12 / Chapter 1.4.1. --- Chromosomal Gains / Chapter 1.4.2. --- Chromosome Losses / Chapter 1.5. --- Epigenetic Changes --- p.18 / Chapter 1.6. --- Aims of Thesis --- p.20 / Chapter Chapter 2 --- Materials and Methods --- p.22 / Chapter 2.1. --- Materials --- p.23 / Chapter 2.1.1. --- Culture of Cell Lines / Chapter 2.1.2. --- Preparation of Normal Human Metaphase / Chapter 2.1.3. --- DNA Extraction from Cell Lines / Chapter 2.1.4. --- DNA Extraction from Tissues / Chapter 2.1.5. --- DNA Extraction from Blood / Chapter 2.1.6. --- Nick Translation / Chapter 2.1.7. --- Dot Blot / Chapter 2.1.8. --- Probe Preparation / Chapter 2.1.9. --- Fluorochrome-conjugated antibodies / Chapter 2.1.10. --- Fluorescence Microscopy and Image Analysis / Chapter 2.1.11. --- Primer Labeling / Chapter 2.1.12. --- Polymerase Chain Reaction / Chapter 2.1.13. --- Gel Preparation / Chapter 2.1.14. --- Gel Electrophoresis / Chapter 2.2. --- Sample --- p.28 / Chapter 2.2.1. --- Patients / Chapter 2.2.2. --- Cell Lines / Chapter 2.3. --- Comparative Genomic Hybridization --- p.30 / Chapter 2.3.1. --- Method / Chapter 2.3.1.1. --- Preparation of Normal Human Metaphase / Chapter 2.3.1.2. --- DNA Extraction / Chapter 2.3.1.3. --- Nick Translation / Chapter 2.3.1.4. --- Labeling Efficiency / Chapter 2.3.1.5. --- Probe Preparation / Chapter 2.3.1.6. --- Slide Preparation / Chapter 2.3.1.7. --- Hybridization / Chapter 2.3.1.8. --- Post Hybridization Wash / Chapter 2.3.1.9. --- Image Capturing and Analysis / Chapter 2.3.1.10. --- Control Experiment / Chapter 2.4. --- Microsatellite Analysis --- p.46 / Chapter 2.4.1. --- Method / Chapter 2.4.1.1. --- Fluorescent-Labeled Polymorphic Markers / Chapter 2.4.1.1.1. --- Polymerase Chain Reaction / Chapter 2.4.1.1.2. --- Gel Preparation / Chapter 2.4.1.1.3. --- Gel Electrophoresis / Chapter 2.4.1.1.4. --- Data Analysis / Chapter 2.4.1.2. --- Radioisotope-Labeled Polymorphic Markers / Chapter 2.4.1.2.1. --- Primer Labeling / Chapter 2.4.1.2.2. --- Polymerase Chain Reaction / Chapter 2.4.1.2.3. --- Gel Preparation / Chapter 2.4.1.2.4. --- Gel Electrophoresis / Chapter 2.4.1.2.5. --- Autoradiography and Data Analysis / Chapter 3. --- Chapter 3 Genetic Imbalances on Chromosome 1 --- p.55 / Chapter 3.1. --- Introduction --- p.56 / Chapter 3.2. --- Methods --- p.57 / Chapter 3.2.1. --- Patients and Cell Lines / Chapter 3.2.2. --- CGH / Chapter 3.2.3. --- MSA with Fluorescent-labeled Polymorphic Markers / Chapter 3.2.4. --- Refinement of lp36 loss / Chapter 3.2.5. --- Investigation of Homozygous Deletion in lp36 / Chapter 3.3. --- Results --- p.63 / Chapter 3.3.1. --- CGH / Chapter 3.3.2. --- MSA on Primary HCC Cases / Chapter 3.3.3. --- Refinement of lp36 loss / Chapter 3.3.4. --- Investigation of Homozygous Deletion in lp36 / Chapter 3.3.5. --- CGH vs MSA / Chapter 3.4. --- Discussion --- p.74 / Chapter 4. --- Chapter 4 Genetic Imbalances on Chromosome 4q --- p.78 / Chapter 4.1. --- Introduction --- p.79 / Chapter 4.2. --- Methods --- p.82 / Chapter 4.2.1. --- Patients and Cell Lines / Chapter 4.2.2. --- CGH / Chapter 4.2.3. --- MSA with Radioisotope-labeled Polymorphic Markers / Chapter 4.3. --- Results --- p.86 / Chapter 4.3.1. --- CGH / Chapter 4.3.2. --- MSA / Chapter 4.3.2.1. --- MSA on Primary HCC cases / Chapter 4.3.2.2. --- MSA on In-house developed HCC cell lines / Chapter 4.3.2.3. --- Combined MSA Results / Chapter 4.4. --- Discussion --- p.94 / Chapter 5. --- Chapter 5 Proposed Future Studies --- p.99 / Chapter 5.1. --- "Microarray Analysis," --- p.101 / Chapter 5.2. --- Functional Studies --- p.102 / Chapter 6. --- Bibliography --- p.104
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Genetic alterations in doxorubicin resistant hepatocellular carcinoma cells: a combined spectral karyotyping, positional expression profiling and candidate genes study.January 2004 (has links)
Hu Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 95-122). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract (in English) --- p.ii / Abstract (in Chinese) --- p.iv / Table of contents --- p.vi / List of figures --- p.x / List of tables --- p.xi / Abbreviations --- p.vii / Chapter CHAPTER ONE: --- INTRODUCATION --- p.1 / Chapter 1.1 --- Hepatocellular Carcinoma --- p.2 / Chapter 1.1.1. --- Epidemiology of HCC --- p.2 / Chapter 1.1.2. --- The major risk factors --- p.2 / Chapter 1.1.3. --- Management of HCC --- p.3 / Chapter 1.2 --- Mechanisms of multidrug resistance (MDR) in cancer cells --- p.4 / Chapter 1.2.1. --- Major mechanisms in reduced drug accumulation --- p.5 / Chapter 1.2.1.1. --- P-glycoprotein (P-gp) --- p.6 / Chapter 1.2.1.2. --- Multidrug Resistance-associated Protein (MRP) --- p.7 / Chapter 1.2.1.3. --- Other effluxes --- p.8 / Chapter 1.2.2. --- Inhibition of apoptotic signaling pathways --- p.11 / Chapter 1.2.2.1. --- TP53 and multidrug resistance --- p.11 / Chapter 1.2.2.2. --- Anti-oncogene PTEN and drug resistance --- p.13 / Chapter 1.2.2.3. --- Influence of BCL2 family on drug resistance --- p.14 / Chapter 1.3 --- The chemotherapeutic agent of doxorubicin --- p.15 / Chapter 1.4 --- Aims of study --- p.18 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.20 / Chapter 2.1 --- Cell culture --- p.21 / Chapter 2.1.1 --- Cell lines and cell culture --- p.21 / Chapter 2.1.2 --- Subculture --- p.23 / Chapter 2.1.3 --- Cryopreservation --- p.23 / Chapter 2.1.4 --- Recovery of cryopreserved culture --- p.24 / Chapter 2.1.5 --- Cell number counting --- p.24 / Chapter 2.2 --- MTT experiments --- p.26 / Chapter 2.2.1 --- Determination of cell seeding density --- p.26 / Chapter 2.2.2 --- Cytotoxic assay --- p.27 / Chapter 2.3 --- Spectral Karytyping (SKY) --- p.27 / Chapter 2.3.1 --- Pretreatment of chromosome slides for SKY --- p.28 / Chapter 2.3.2 --- Hybridization --- p.28 / Chapter 2.3.3 --- Detection --- p.29 / Chapter 2.4 --- Positional expression profiling --- p.30 / Chapter 2.4.1 --- RNA extraction --- p.32 / Chapter 2.4.2 --- Reverse transcription and cDNA labling --- p.34 / Chapter 2.4.3 --- Probe purification and hybridization --- p.34 / Chapter 2.4.4 --- Image acquisition and data analysis --- p.35 / Chapter 2. 5 --- Quantitative RT-PCR --- p.37 / Chapter 2.5.1 --- RNA extraction --- p.37 / Chapter 2.5.2 --- Primer design --- p.37 / Chapter 2.5.3 --- Reverse transcription --- p.37 / Chapter 2.5.4 --- Quantitative PCR --- p.39 / Chapter 2.6. --- Statistical analysis --- p.40 / Chapter CHAPTER 3 --- RESULTS --- p.43 / Introduction --- p.44 / Chapter 3.1 --- Doxorubicin resistance in HCC cell lines --- p.44 / Chapter 3.2 --- Candidate drug resistance genes --- p.56 / Chapter 3.3 --- The roles of chromosomal instability --- p.58 / Chapter 3.4 --- Candidate resistance genes identified in chromosome 10 --- p.69 / Chapter CHAPTER 4 --- DISCUSSION --- p.75 / Introduction --- p.76 / Chapter 4.1 --- In vitro cell models facilitate drug resistance investigations --- p.11 / Chapter 4.2 --- Aneuploidy and DX resistance --- p.78 / Chapter 4.3 --- The role of known resistance genes on chromosome 10 --- p.79 / Chapter 4.4 --- Identification of novel DX resistance genes on chromosome 10 --- p.80 / Chapter 4.5 --- Common drug resistance genes --- p.83 / Chapter 4.5.1. --- The roles of classical drug resistance --- p.85 / Chapter 4.5.2. --- Inhibition of apoptosis and deregulation of cell cycle --- p.86 / Chapter CHAPTER 5 --- PROPOSED FUTURE STUDIES --- p.90 / Chapter 5.1. --- Validate significant in vitro findings by clinical trials --- p.91 / Chapter 5.2. --- Molecular mechanisms in inactivation of ECHS1 in resistant cells --- p.92 / Chapter 5.3. --- Future utilization of cDNA microarray data --- p.93 / REFERENCES --- p.95 / PUBLICATION --- p.122
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Effects of Agrimonia pilosa Ledeb. on hepatocarcinogenesis in rats.January 2003 (has links)
Li Qian. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 102-117). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.vi / List of Abbreviations --- p.ix / List of tables and figures --- p.ix / Content --- p.x / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Traditional Chinese Medicine: Agrimony --- p.1 / Chapter 1.2 --- Hepatocellular carcinoma (HCC) and its risk factors --- p.4 / Chapter 1.3 --- Basic concepts relevant to cancer prevention --- p.6 / Chapter 1.3.1 --- Multistage process of carcinogenesis --- p.6 / Chapter 1.3.2 --- Chemical carcinogenesis --- p.7 / Chapter 1.3.3 --- Possible chemopreventive strategies --- p.8 / Chapter 1.3.4 --- Phase I and phase II systems in chemical carcinogenesis --- p.10 / Chapter Chapter 2 --- Materials and methods --- p.12 / Chapter 2.1 --- Preparation of aqueous extract of Agrimonia pilosa --- p.12 / Chapter 2.2 --- In vivo study --- p.13 / Chapter 2.2.1 --- Animal model for hepatocarcinogenesis --- p.13 / Chapter 2.2.1.1 --- Chemical carcinogens --- p.13 / Chapter 2.2.1.2 --- Animals --- p.16 / Chapter 2.2.1.3 --- Animal treatment and sacrifice --- p.17 / Chapter 2.2.2 --- Histological and immunohistochemical study --- p.20 / Chapter 2.2.3 --- Preparation of liver homogenates and microsomes from rat --- p.23 / Chapter 2.2.4 --- Determination of protein concentration --- p.24 / Chapter 2.2.5 --- COX-2 Activity Assay --- p.25 / Chapter 2.2.6 --- Cytochrome P450 2E1 Assay --- p.26 / Chapter 2.2.7 --- Spectrophotometry Assay for GST --- p.28 / Chapter 2.2.8 --- Isolation of total RNA from liver homogenate --- p.29 / Chapter 2.2.9 --- Semi-quantitative RT-PCR analysis --- p.32 / Chapter 2.3 --- In vitro study --- p.36 / Chapter 2.3.1 --- Cell cultures --- p.36 / Chapter 2.3.2 --- Cytotoxicity assay - Neutral Red Assay --- p.38 / Chapter 2.3.3 --- Cell cycle distribution analysis by flow cytometry --- p.39 / Chapter 2.3.4 --- DNA fragmentation --- p.40 / Chapter Chapter 3 --- Results --- p.43 / Chapter 3.1 --- In vivo study --- p.43 / Chapter 3.1.1 --- Body weight and relative liver weight --- p.43 / Chapter 3.1.2 --- Gross Morphological changes --- p.46 / Chapter 3.1.3 --- Hematoxylin & Eosin (H&E) staining for histological detection --- p.50 / Chapter 3.1.4 --- Effect of AP on DEN-CCl4-induced GST-P positive foci formation and GST-P mRNA expression --- p.60 / Chapter 3.1.5 --- Effects of AP on COX-2 --- p.72 / Chapter 3.1.6 --- Effects of AP on phase I and phase II enzymes --- p.76 / Chapter 3.2 --- In vitro study --- p.80 / Chapter 3.2.1 --- Effects of AP on proliferation of H4IIE cells detected by Neutral Red Assay --- p.80 / Chapter 3.2.2 --- Assessment of cell cycle distribution by flow cytometry --- p.82 / Chapter 3.2.3 --- DNA Fragmentation Assay --- p.88 / Chapter Chapter 4 --- Discussion --- p.90 / Chapter 4.1 --- In vivo study --- p.90 / Chapter 4.1.1 --- Morphological changes during the induction of hepatocarcinogenesis --- p.90 / Chapter 4.1.2 --- Effects of AP on GST-P foci and its mRNA --- p.91 / Chapter 4.1.3 --- Effects of AP on COX-2 enzyme activity and mRNA expression --- p.93 / Chapter 4.1.4 --- Modulation effects of AP on CYP2E1 and GST enzyme activity --- p.95 / Chapter 4.2 --- In vitro study: effects of AP on cancer cell proliferation --- p.97 / Chapter 4.3 --- Summary --- p.99 / References --- p.102
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The functional characterization of 1,3,5-trihydroxy-13,13-dimethyl-2H-pyran [7,6-b] xanthone in hepatocellular carcinoma: targeting heat shock protein 27 to mediate mitochondrial apoptosis.January 2012 (has links)
研究背景: / 肝癌是全球常見的惡性腫瘤之一,世界上每年大約有50萬死亡病例,並且呈逐年上升之勢, 是全球第3位的腫瘤死亡原因。慢性乙型和丙型肝炎病毒感染是肝癌的主要成因。肝癌惡性程度高、預後差,並且目前的治療手段非常有限,術後易復發和轉移,迄今尚無正式獲准有效治療藥物。現階段,治療肝癌的主要方法是手術切除,但是隨之引起的併發症以及較高的復發機率嚴重影響了治療的療效,大大降低肝癌病人的存活期。 / 研究目的: / 分析TDP對肝癌細胞和肝腫瘤旁細胞生長的影響;分析TDP抑癌的分子靶標蛋白及其分子機理;驗證TDP對肝癌動物模型的抑制效果。開發一種新型有效的肝癌治療藥物。 / 研究方法: / 首先用MTT法從102種來源於嶺南山竹子的純複合物中分離出了TDP,它是一種甾醇類化合物。採用MTT法檢測TDP對腫瘤細胞生長的影響;流式細胞實驗驗證TDP能否引起腫瘤細胞的凋亡;採用蛋白組學和質譜分析找出TDP抑癌的分子靶標;進一步的蛋白功能增加和缺失實驗證明Hsp27的功能和作用;生物資訊學驗證HSP27和TDP的作用結果;最後利用動物模型驗證TDP對肝腫瘤的治療效果。 / 結果: / TDP不但能效率極高的抑制肝癌細胞的生長而且可以大量誘發肝癌細胞的凋亡,而對正常的肝癌旁細胞沒有影響。二維電泳以及質譜分析TDP處理的肝癌細胞對比DMSO處理的肝癌細胞發現了具有不同表達水準的18種蛋白,Hsp27是其中一個在TDP誘導下調變化倍數較大並且與細胞凋亡有密切關係的蛋白,Hsp27的過表達以及Knock-down都充分驗證了TDP通過調節Hsp27的表達參與了依賴於caspase的線粒體凋亡途徑,在Western Blotting以及RT-PCR中得到了充分的驗證。生物資訊學預測TDP可以與Hsp27結合,實驗結果表明TDP可以誘導Hsp27的聚集並導致功能喪失。動物實驗腫瘤生長結果以及免疫組化結果證明,TDP可以在很大程度上對肝癌有抑製作用。 / 結論: / 本研究首次表明,TDP如果不是完全的,最起碼也是部分通過誘導依賴於caspase的線粒體凋亡的途徑來抑制肝癌細胞的增值和分化, 具有明顯的抗腫瘤的功效,特別是對Hsp27高表達的腫瘤細胞有比較明顯的作用,是一種值得繼續深入研究的有較高潛在價值的藥物。 / Background: / Hepatocellular carcinoma (HCC), the most common primary hepatic malignancy, is a global public health problem that accounts for approximately 500,000 deaths annually. Chronic hepatitis B and hepatitis C infections are the major risk factors for the development of HCC. Due to the high rate of these infections, the incidence of HCC remains alarmingly high globally. Although great advances have been made in HCC treatment, poor prognosis and high risk of recurrence have been major challenges to patients. Currently, surgical resection is the main treatment option for HCC patients; however, complications arising from surgery can threaten its therapeutic effect and patients’ survival. / Objectives: / To characterize the functions of 1,3,5-trihydroxy-13,13-dimethyl-2H-pyran [7,6-b]Xanthone (TDP) in cell proliferation of HepG2 cells; to discover the molecular target genes and elucidate the underlying molecular mechanism of TDP; to examine the in vivo function of TDP in a nude mouse tumor model of HCC. Finally, to investigate TDP’s potential as an anti-HCC drug candidate. / Methods and Results: / In this study, we discovered that TDP, isolated from the Chinese medicinal herb, Garcinia oblongifolia, strongly inhibited cell growth and induced caspase-dependent mitochondrial apoptosis in HCC, as evidenced from MTT assay and flow cytometry analysis. Two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomics were applied to find the molecular targets of TDP in HCC cells, and eighteen proteins were identified with altered expression, with Hsp27 protein being one of the proteins most significantly down-regulated by TDP. Further Hsp27-siRNA knockdown and Lenti-Hsp27 overexpression studies found that Hsp27 was involved in TDP induced mitochondrial apoptosis, with bioinformatics predictions and biological results revealing that TDP might cause Hsp27 protein form dimer and consequent degradation via the ubiquitin-proteasome system. Finally, subcutaneously injecting cancer cells with Hsp27 expression vector into the dorsal flank of nude mice tumor model also demonstrated the suppressive effect of TDP on HCC. / Conclusions: / In summary, our study discovered that TDP, a natural xanthone, was a potent inhibitor of Hsp27 in HCC. TDP inhibited cell growth and induced apoptosis by inducing Hsp27 degradation, which stimulated mitochondrial cytochrome C release which resultantly activated caspase-3 and caspase-9. These data combined with the results of the animal model strongly supported TDP’s potential as a novel anti-cancer drug candidate, especially for cancers with an abnormally high expression of Hsp27. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Fu, Weiming. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 111-151). / Abstract also in Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iiv / Acknowledgment --- p.vi / Publications --- p.viii / List of Contents --- p.ix / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.1.1 --- Overview of HCC --- p.1 / Chapter 1.1.2 --- Epidemiology of HCC in China and Hong Kong --- p.3 / Chapter 1.2 --- Etiology of HCC --- p.7 / Chapter 1.2.1 --- Cirrhosis --- p.8 / Chapter 1.2.2 --- HBV infection --- p.9 / Chapter 1.2.3 --- HCV infection --- p.10 / Chapter 1.2.4 --- Viral Co-Infection --- p.11 / Chapter 1.2.5 --- Fatty Liver Disease and Cryptogenic Cirrhosis --- p.12 / Chapter 1.2.6 --- Alcohol --- p.13 / Chapter 1.2.7 --- Iron --- p.13 / Chapter 1.2.8 --- Aflatoxin --- p.14 / Chapter 1.2.9 --- Others --- p.14 / Chapter 1.3 --- Diagnosis of HCC --- p.14 / Chapter 1.4 --- Prognosis of HCC --- p.17 / Chapter 1.5 --- Treatment of HCC --- p.19 / Chapter 1.5.1. --- Early stage --- p.19 / Chapter 1.5.2. --- Intermediate and advanced stage --- p.24 / Chapter 1.5.3. --- Terminal stage --- p.28 / Chapter 1.6 --- Signaling pathways in HCC --- p.28 / Chapter 1.6.1 --- Proliferation signaling pathways --- p.29 / Chapter 1.6.2 --- Signaling pathways frequently dysregulated in HCC --- p.30 / Chapter 1.6.3 --- Pathways involved in liver development and cell differentiation --- p.34 / Chapter 1.6.4 --- Inflammation pathways involved in hepatocarcinogenesis --- p.35 / Chapter 1.6.5 --- Pathways involved in neoangiogenesis --- p.37 / Chapter 1.6.6 --- The P53 tumor suppressor --- p.38 / Chapter 1.6.7 --- Heat shock proteins in HCC --- p.39 / Chapter 1.7 --- The roles of microRNAs in liver cancer progression --- p.42 / Chapter 1.8 --- TCM in the treatment of HCC --- p.45 / Chapter 1.8.1 --- Introduction --- p.45 / Chapter 1.8.2 --- Garcinia --- p.49 / Chapter 1.9 --- Objectives of the study --- p.51 / Chapter Chapter 2 --- Materials and Methods --- p.52 / Chapter 2.1 --- Preparation of the pure compounds --- p.52 / Chapter 2.2 --- Liver cell lines and tissue culture --- p.52 / Chapter 2.3 --- Human tissue samples --- p.52 / Chapter 2.4 --- Cell viability assessment with MTT assay --- p.53 / Chapter 2.5 --- Apoptosis analysis --- p.53 / Chapter 2.6 --- Two-dimensional electrophoresis (2-DE), protein visualization and image analysis --- p.54 / Chapter 2.6.1 --- Materials --- p.54 / Chapter 2.6.2 --- Protein extraction --- p.54 / Chapter 2.6.3. --- 2-DE protein profiling --- p.55 / Chapter 2.6.4. --- Gel staining and image analysis --- p.55 / Chapter 2.6.5. --- In-gel protein digestion with trypsin --- p.56 / Chapter 2.6.6. --- MALDI-TOF mass spectrometric analysis --- p.56 / Chapter 2.6.7. --- Database search --- p.57 / Chapter 2.7.1 --- Sample preparation --- p.58 / Chapter 2.7.2 --- SDS-PAGE --- p.58 / Chapter 2.7.3 --- Protein transfer --- p.58 / Chapter 2.7.4 --- Blocking --- p.59 / Chapter 2.7.5 --- Incubation with primary and secondary antibodies --- p.59 / Chapter 2.7.6 --- Proteins Visualization --- p.59 / Chapter 2.8 --- Real-time PCR --- p.60 / Chapter 2.9 --- Vector construction and lentivirus production --- p.61 / Chapter 2.9.1 --- Lenti-vector construction for Hsp27 expression --- p.61 / Chapter 2.9.2 --- Lentivirus production --- p.62 / Chapter 2.9.3 --- Lentivirus infection --- p.63 / Chapter 2.10 --- SiRNAs transfection. --- p.63 / Chapter 2.11 --- Identification of potential protein targets for TDP --- p.64 / Chapter 2.12 --- In Vivo Tumorigenesis --- p.64 / Chapter 2.13 --- Assay of chaperone activity of Hsp27 using lysozyme as substrate --- p.65 / Chapter 2.14 --- Mitochondria and cytosolic proteins preparation --- p.66 / Chapter 2.15 --- Immunohistochemistry (IHC) --- p.67 / Chapter 2.15.1 --- Preparation of paraffin tissue sections --- p.67 / Chapter 2.15.2 --- Immunostaining --- p.67 / Chapter 2.16 --- Methodology of this study --- p.68 / Chapter 2.17 --- Statistical analysis --- p.68 / Chapter CHAPTER 3 --- Results --- p.69 / Chapter 3.1 --- Introduction --- p.69 / Chapter 3.2 --- TDP significantly suppressed cell growth and induced apoptosis in HCC cells. --- p.69 / Chapter 3.2.1 --- TDP was identified from 102 pure compounds by using MTT assay --- p.69 / Chapter 3.2.2 --- TDP significantly suppressed HCC cell growth --- p.73 / Chapter 3.2.3 --- TDP induced the apoptosis of HCC cells --- p.74 / Chapter 3.3 --- Study of the molecular mechanism of TDP on HCC --- p.76 / Chapter 3.3.1 --- The comparative proteomic profiling --- p.76 / Chapter 3.3.2 --- Hsp27 was one of the molecular targets of TDP in HepG2 cells. --- p.80 / Chapter 3.3.3 --- TDP induced apoptosis through the caspase-dependent mitochondrial pathway. --- p.82 / Chapter 3.3.4 --- Hsp27 involved in the mitochondrial apoptosis induced by TDP --- p.84 / Chapter 3.3.5 --- Enforced Hsp27 overexpression rescued the mitochondrial apoptosis induced by TDP in HepG2 cells --- p.87 / Chapter 3.3.6 --- The possible regulatory signaling by TDP --- p.91 / Chapter 3.4 --- TDP directly targeted Hsp27 and destroyed its chaperone action --- p.92 / Chapter 3.5 --- Degradation of Hsp27 aggregation stimulated by TDP was mediated by ubiquitin-proteasome system (UPS) pathway --- p.96 / Chapter 3.6 --- Nude mice model demonstrated the suppressive effect of TDP on HCC --- p.97 / Chapter Chapter 4 --- Discussion and Conclusions --- p.100 / Chapter 4.1 --- Discussion --- p.100 / Chapter 4.2 --- Conclusion --- p.110 / Reference --- p.111
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Identification and characterization of pathogenetic events in the progression of human hepatocellular carcinoma. / CUHK electronic theses & dissertations collectionJanuary 2005 (has links)
Hepatocellular carcinoma (HCC) is a highly malignant tumor that is prevalent in Southeast Asia and China, where hepatitis B viral (HBV) infection is the main etiologic factor. Despite a high incidence of HCC developing in patients with HBV-induced liver cirrhosis, the molecular events underlying the malignant liver progression remain largely unclear. In an effort to characterize the genetic abnormalities involved in the HBV-related liver carcinogenesis, genome-wide exploration by metaphase comparative genomic hybridization (CGH) was performed on 100 cirrhotic HCC tumors that were derived from chronic hepatitis B carriers. CGH analysis indicated chromosomal instability in both early and advanced stage tumors where common genomic copy gains on 1q, 8q and 17q, and deletions on 4q, 8p, 13q, 16q and 17p found in both groups are suggestive of early events in hepatocarcinogenesis. Nevertheless, a combined univariate and multivariate statistical analyses highlighted for the first time preferential regional 3q26-q28, 7q21-q22 and 7q34-q36 gains in association with advanced stage HCC. The novel aberrant gains identified here thus formed basis for further mapping analysis for causative genes related to HCC progression in this thesis. / Near 50% of the advanced stage HCC manifested copy gains of chr 7q21-q22. High resolution mapping analysis by cDNA microarray-based CGH nominated 13 amplified candidates within the region 7q21-q22 Analysis on the mRNA expresson levels of these genes in a cohort of primary HCC compared to paired adjacent non-tumorous liver tissues by quantitative RT-PCR (qRT-PCR) indicated the up-regulation of the PFTK1 (PFTAIRE protein kinase 1) gene as the only candidate that demonstrated a close association with advanced metastatic tumors. The effects of PFTK1 on cell proliferation, migration and invasive phenotypes were further studied to substantiate its role in HCC progression. Upon gene suppression of PFTK1 in vitro by RNA interference (RNAi), a significant reduction in chemotactic migration, cellular invasion and an inhibition on cell motility were indicated, albeit cell proliferation remained unaffected. / Sub-cellular localization study of translated PFTK1 protein indicated protein localization in both the nucleus and cytoplasm. This has led to the further investigations of potential PFTK1 function at both the transcriptional and protein levels. (Abstract shortened by UMI.) / Sy Ming Hui. / "July 2005." / Advisers: Winnie Yeo; Nathalie Wong. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3571. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 124-139). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.
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Differential expressed microRNA in the development of hepatocellular carcinoma. / CUHK electronic theses & dissertations collectionJanuary 2008 (has links)
In summary, the genome-wide miRNA analyses on HCC tumors, adjacent non-malignant livers and cell lines revealed distinct differential miRNA expressions. In particular, the findings of deregulated miR-223 and miR-222 underscore the potential role for these microRNAs in the development of HCC. / In the functional examinations of miR-222, inhibition of miR-222 expression in Hep3B and HKCI-9 exerted no effect on cell viability. However, significant retardations on cell migration were observed in both Hep3B (64.5%, p=0.008) and HKCI-9 (52.5%, p=0.048). In Hep3B cells, functional knockdown of miR-222 was further shown to impede filopodia formation (p=0.0273). Coupling expression profiling from functional knockdown of miR-222 in Hep3B and HKCI-9 with pathway analysis, a number of miR-222 modulated pathways was suggested. Examination of such pathways, AKT and JAK/STAT, by Western blot analysis suggested profound decrease of total AKT and STAT3 protein in both Hep3B and HKCI-9. A corresponding diminution of phosphorylated AKT was also shown in both cell lines. In the examination of JAK/STAT pathway, reductions of phosphorylated STAT3 proteins were demonstrated in Hep3B and HKCI-9 following functional knockdown of miR-222. Parallel quantitative RT-PCR analysis did not suggest transcriptional changes of AKT and STAT3 mRNA between miR-222 inhibited cells and mock controls in Hep3B and HKCI-9. This in turn would be suggestive of a post-transcriptional repression of AKT and STAT3 proteins by miR-222 knockdown. Based on the functional characterization of miR-222, it would suggest the likelihood of miR-222 induction on HCC cell motility through modulation of the AKT and JAK/STAT signalling pathways. / MicroRNAs (miRNAs) are an abundant class of small, 19-25 nucleotides, non-coding RNAs with significant roles in transcriptional silencing and translational suppression. Recent studies have emphasized on a causative link between miRNA deregulations and cancer development. However, such information remains minimal in Hepatocellular Carcinoma (HCC). In an effort to characterize differentially expressed miRNAs in HCC development, global expression analyses on HCC tumors, paired adjacent non-malignant livers and HCC cell fines were carried out. Distinct miRNA expression pattern that was able to distinguish HCC tumors from non-malignant cirrhotic livers was suggested. Based on a comprehensive screening, 96 miRNAs showed differential expressions in HCC tumors, within which over 60% of miRNAs displayed increased expressions. / Six top ranked differentially expressed miRNAs, namely down-regulated miR-223, miR-126 and miR-122a, and up-regulated miR-222, miR-221 and miR-31 were subjected to further Northern blot validations. Amongst these verified candidates, miR-223 and miR-222 showed the most consistent expression changes that allowed unequivocal differentiation between HCC and non-tumoral liver (p≤0.002). The potential functional roles of miR-223 and miR-222 were subsequently investigated. Ectopic expression of miR-223 in 3 HCC cell fines, Hep3B, HKCI-C3 and HKCI-10, revealed a consistent growth inhibitory effect of 21-44% (p≤0.01). In an attempt to define potential downstream targets of miR-223, an integrative analysis of overexpressed genes from mRNA array with in-silico predictions was utilized. This approach allowed streamline of 386 targets to a candidate gene, Stathmin1 (STMN1). A significant inverse correlation between STMN1 mRNA and miR-223 expressions was demonstrated (p=0.006). At the protein level, restoration of miR-223 expressions in HCC cell lines resulted in substantial reduction of STMN1. Furthermore, miR-223 could repress the luciferase activity in reporter construct containing the putative recognition site at the STMN1 3'UTR. / Wong, Wing Lei. / Adviser: Nathalie Wong. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3450. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 163-171). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.
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Identification of serum biomarkers for hepatocellular carcinoma by glycoproteomic analysis. / CUHK electronic theses & dissertations collectionJanuary 2007 (has links)
Aim. In this study, we attempted to identify HCC-specific serum glycoproteins by using glycoproteomic technologies. We targeted at finding glycoforms with aberrant alpha-2,6-sialylation and alpha-1,6-fucosylation in sera of HCC patients. These glycoforms may have potentials to be used as tumor marker(s) in the diagnosis of HCC. / Among the validated differential glycoproteins, Hp was the only glycoprotein, glycoforms of which were found to be significantly up-regulated in the HCC group when examining both the sialylated glycoprotein profiles and the fucosylated glycoprotein profiles. This glycoprotein was selected for further investigation. In the independent validation group, increased serum levels of Hp (total), alpha-2,6 sialylated Hp and alpha-1,6 fucosylated Hp was observed in the HCC patients. A unique pattern of Hp glycoforms comprising both hypersialylated fucosylated and hyposialytated fucosylated species was found in the HCC patients. Serum concentrations of these glycoforms were significantly higher in the HCC patients with advanced tumor, suggesting their tumor-specific nature. Besides, we have performed quantitative profiling of N-glycans of serum Hp in the HCC patients, CLD patients and normal subjects, and have attempted to identify HCC-associated N-glycans for HCC diagnosis. Combined used of serum alpha-fetoprotein, serum Hp and its N-glycans, we could achieve 84% sensitivity at 100% and 93% specificities when distinguishing the HCC patients from the CLD patients and from the normal subjects, respectively. / Background. Hepatocellular carcinoma (HCC) often arises in patients with coexisting chronic liver disease (CLD). Since alpha-fetoprotein, a conventional biomarker, may also be raised in patients with uncomplicated CLD, the use of alpha-fetoprotein in early detection of HCC is limited. Identification of additional biomarkers may improve early detection. Previous studies have shown that levels of alpha-2,6-sialyltransferase and alpha-1,6-fucosyltransferase change in liver cancer, leading to aberrant glycosylations on some serum proteins. / Conclusion. By undertaking glycoproteomic approach, we have identified a panel of potential biomarkers for diagnosis of HCC. These biomarkers were useful for classifying among normal healthy subjects, CLD patients, patients with early HCC and patients with advanced HCC. Some of them were validated with the independent cases. Finally, we have identified a unique pattern of Hp glycoforms comprising both hypersialylated fucosylated and hyposialylated fucosylated species in the HCC patients. Serum Hp and its N-glycans have been shown to have potential values for aiding the diagnosis of HCC. / Methodology. There are four parts in this study. The first part is "method development". A method for obtaining quantitative profiles of serum glycoproteins with alpha-2,6-sialylation or alpha-1,6-fucosylation was developed by combined use of lectin affinity chromatography, two-dimensional polyacrylamide gel electrophoresis and enzyme-linked lectin assay. The second part is "biomarker discovery". The quantitative profiles of the serum glycoproteins from 20 HCC patients and 10 CLD patients (control) were compared by bioinformatic approaches to identify potential biomarkers for diagnosis of HCC. The protein identities of the potential targets were obtained by using MALDI-TOF/TOF mass spectrometry. The third part is "validation". An independent set of serum samples from 40 HCC patients, 30 CLD patients and 20 normal subjects was used to evaluate the diagnostic values of the potential biomarkers. The last part of this study was aimed to identify HCC-associated N-glycans on one of potential biomarkers found, and examine their values in diagnosis of HCC. / Result. When analyzing alpha-2,6-sialylated glycoproteins, 53 glycoprotein spots were significantly different between the HCC and CLD groups, of which 44 spots belonged to 13 glycoproteins. Bioinformatic analyses revealed that these differential sialoglycoprotein profiles contained valuable information for differentiating the HCC patients from CLD patients, and classifying between early HCC and advanced HCC patients. When analyzing alpha-1,6-fucosylated proteins, 11 glycoprotein spots were significant different between the two study groups, of which 8 spots belonged to 1 glycoprotein. Majority of the protein identities were successfully obtained by MALDI-TOF/TOF mass spectrometry. Among the differential glycoproteins, we have identified a subgroup with a unique pattern of glycosylation. These glycoproteins were characterized by the presence of hypersialylated and fucosylated glycoforms. The differential patterns and the diagnostic values of some of these serum glycoproteins were confirmed in the independent validation group by measuring their serum levels with immunoassays. The results of the logistic regression analyses suggest that complement factor B and haptoglobin (Hp) can be used in combination with alpha-fetoprotein to improve the diagnosis of HCC. / Ang, Ling. / Adviser: Tereng Chuen Wai Poon. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0947. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 215-238). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / School code: 1307.
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Functional characterization of novel HBV subgenotypes/mutations associated with increased risk for hepatocellular carcinoma (HCC). / CUHK electronic theses & dissertations collectionJanuary 2009 (has links)
After alignment of 300 HBV sequences randomly downloaded from GenBank, we found that the frequency of A1762T and G1764A mutations in genotype C was found as high as 64%, while 34% was found for other genotypes (A, B, D to H). Besides, recent clinical studies have also shown that A1762T/G1764A mutations occur frequently in HCC patients with genotype B infection (81%, 30 of 37 patients), but were relatively lower in asymptomatic carriers (43%, 22 of 51 patients). These indicate that the contribution of A1762T/G1764A mutations to liver cancer might not be limited to genotype C. As the double mutations are present within the region of HBV Enhancer II/Basal core promoter (BCP) and cause residue substitution of HBx (Lys130Met and Val131Ile); therefore, their effects on the promoter and HBx activities were examined. / Chronic infection of hepatitis B virus (HBV) increases the risk of hepatocellular carcinoma (HCC) by more than 100-fold. However, the underlying molecular mechanism of this process is not fully understood. Several recent studies have shown that A1762T and G1764A mutations of HBV were associated with the aggressiveness of liver disease, in which inactive carriers would develop active hepatitis, and eventually liver cirrhosis and HCC. In Asia, genotypes B and C are the predominant genotypes of HBV infections. Our longitudinal five-year follow-up study of 426 chronic hepatitis B patients in Hong Kong found that the genotype C HBV (normally with A1762T/G1764A mutations) was closely associated with higher risk of HCC than genotype B HBV (non-frequent mutations with A1762T/G1764A). / In this study, systemic site-directed mutagenesis studies, promoter assays, replication capacity assays and overexpression of HBx assays were carried out to demonstrate the molecular mechanisms of these mutations for the increases risk of HCC. Three conclusions were drawn from this study. (1) A1762T and/or G1764A mutations of HBV could reduce BCP activities in a synergistic manner with 1764A contributing more. Reversed T1762A and/or A1764G mutations increase the BCP activities also in a synergistic manner with 1764G contributing more; (2) HBx could increase HBV BCP activity, HBV replication and HBsAg expression. The Lys130Met and Val131Ile mutations of HBx could further increase the above abilities while the A1762T/G1764A double mutations in the BCP region could not affect the interaction of HBx and HBV BCP; (3) The G1677T/A1679C and T1706C mutations could increase the BCP activity; The ectopic expression of HBx could further increase the BCP activity while the mutated HBx (130Met and 131Ile) has less effect on these mutated promoters. / Dong, Qingming. / Adviser: Ming-Liang He. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis submitted in: December 2008. / Thesis submitted in: December 2008. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 132-154). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.
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The functional study of HCC-associated mutations on hepatitis B virus. / CUHK electronic theses & dissertations collectionJanuary 2010 (has links)
A case-control study was previously carried out to identify HCC-associated genomic markers on HBV. Some of them are clustered at the preS1 and X promoter regions of HBV genotype B and core promoter of HBV subgenotype Cs. The functional significance of these markers to the virus was investigated in our study. Our result showed that one of those markers, the G1613A mutation on core promoter, can significantly increase the promoter activity in a genotype-dependent manner and the effect is reversible by the A-to-G back mutation. We have established an in vitro full-length HBV genome transfection system and the result suggested that the G1613A mutation suppressed the e antigen (HBeAg) secretion and enhanced virus DNA production by downregulating the precore (preC) mRNA transcription. In consistence to the clinical study, the mutation was associated to serum HBV DNA level higher than 6 log copies/1M in female HBV carriers in a univariate analysis. In addition, we demonstrated that the G1613A mutation is a hot spot mutation situated on the negative regulatory element (NRE) on the core promoter in an alignment analysis. To further investigate the molecular mechanism of the mutation, two unknown protein complexes had been shown to bind on the NRE. They showed different binding affinity to the G1613-wild-type and A1613-mutant NRE sequence. Moreover, we showed that in vitro synthesized RFX1 protein could bind to the mutated NRE probe at a higher affinity than that to wild-type NRE probe. Overall, our result suggests that the G1613A mutation exerts its effect by differential binding to some proteins via the NRE region. Studying the mechanism of the mutations may provide insights to the viral pathogenesis and HBV-associated HCC, which has long been a health burden in Asia-Pacific countries. / Infection of hepatitis B virus (HBV) causes acute and chronic hepatitis and is closely associated with the development of cirrhosis and hepatocellular carcinoma (HCC). Approximately 60-80% of world's HCC is related to HBV, and it is the third most common cause of cancer death in Asia-Pacific region. Almost 400 million people are chronically infected with HBV and one-third was likely to die of complications of cirrhosis, including liver failure and HCC. As there is a shortage of effective curative treatments, detection and prognosis of the risk of cancer development will be essential to improve survival of patients with chronic HBV infection. / Li, Man Shan. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 198-210). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.
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