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.

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.

研究背景和目的 / 肝癌是非常高死亡率的恶性肿瘤之一。由于传统化疗方式的局限性，表观遗传治疗方法可能成为肝癌治疗的替代方法。研究报道ZBP-89诱导肝癌细胞Bak的表达，表观调控是否参与该诱导作用，目前仍然不清楚。 / HDAC3被认为是化疗靶点和肝癌复发的肿瘤标记物。它常常在肝癌组织中高表达，对HDAC3的抑制作用可以增加肝癌的化疗效果。我们的研究表明ZBP-89可以降低肝癌细胞HDAC3的表达，但机制未明。蛋白的翻译后调控是细胞生化过程的重要调节因素。所以，研究调节HDAC3的降低途径对肝癌的发生和复发具有非常重要的研究意义。 / 本研究旨在研究ZBP-89调控Bak表达的表观遗传机制。同时，弄清楚DNA甲基化转移酶和组蛋白去乙酰化酶是否参与ZBP-89对Bak的调控作用，进一步阐明ZBP-89对HDAC3降低通路的机制。 / 方法和结果 / 肝癌病人组织蛋白分析表明，相对于癌旁组织，肝癌组织Bak和ZBP-89蛋白表达降低，而DNMT1和HDAC3表达升高。免疫共沉淀技术显示ZBP-89与HDAC3、 DNMT1结合，但不与HDAC4， DNMT3a和DNMT3b结合。相应地，HDAC3和 DNMT1免疫沉淀分析也显示三者形成免疫复合物。我们在肝癌细胞中过表达ZBP-89，验证它会不会影响HDACs和DNMTs的活性。实验结果表明过表达的ZBP-89抑制HDACs和DNMTs的活性。进一步发现ZBP-89调节的Bak表达可能是通过抑制HDACs活性和维持组蛋白H3和H4乙酰化水平实现的。另一方面，我们同样证明HDAC的抑制剂（HDACi）VPA和TSA可以诱导肝癌细胞Bak表达，此外，siRNA干扰HDAC3的表达同样可以诱导Bak表达。 / 对DNMT1表达的抑制和使用DNMT抑制剂（DNMTi）Zebularine也可以诱导Bak的表达。染色质免疫沉淀结果显示ZBP-89结合于Bak的启动子区域，从-3188bp到-3183bp，从-275到-49。 ZBP-89可以抑制DNMT的活性，那么ZBP-89是否会影响DNA中CpG岛甲基化状态和甲基化结合蛋白（MeCP2）的结合能力，这一点仍需要进一步证实。结果表明ZBP-89可以抑制MeCP2结合基因组DNA。为进一步揭示MeCP2是否由于启动子区域CpG岛去甲基化影响其结合能力，我们采用亚硫酸盐测序方法。测序结果显示ZBP-89过表达可以影响Bak启动子CpG岛的甲基化状态，并促进其去甲基化。 / 腺病毒介导的ZBP-89过表达降低HDAC3表达呈现剂量依赖性，然而HDAC3 的mRNA水平并没有受到ZBP-89的表达。免疫共沉淀方法和蛋白免疫印迹实验用于分析Pin1和HDAC3复合物，磷酸化IκB和HDAC3复合物的结合情况。结果表明Pin1结合HDAC3并促进HDAC3的减少。同时，HDAC3与磷酸的IκB结合并进入蛋白减少途径。 / 构建的mU6-siPin1表达质粒用于敲除肝癌细胞Pin1的表达，方法检测基因表达水平。Pin1的缺失表达阻碍ZBP-89介导的HDAC3降低。在Pin1 敲除细胞系 JB6 C141 Pin1⁻/⁻ 和Pin1过表达细胞系的研究，ZBP-89更加能促进Pin1⁺/⁺细胞中HDAC3减少，而对Pin1⁺/⁺的细胞则没那么明显。由此肯定了Pin1在ZBP-89介导的HDAC3降低中的重要作用。进一步研究发现， IκB激酶 (IKK)抑制剂，CAY10576，能抑制 ZBP-89介导的HDAC3的降低；而SN50, p65/p50人核抑制多肽，则不影响HDAC3的降低。研究结果证明HDAC3的降低依赖IκB通路，而不是NF- κB活性。 / 我们用人肝癌细胞的裸鼠移植瘤模型研究ZBP-89调控Bak表达的表观遗传机制，及其对肝癌的治疗效果。研究结果表明ZBP-89蛋白和组蛋白抑制剂VPA和DNA甲基化抑制zebularine都能抑制肿瘤的生长，并诱导肿瘤组织Bak表达及细胞凋亡。VPA和zebularine联合治疗的效果更好。研究也表明ZBP-89可以在体内降低HDAC3蛋白水平。 / 结论 / 本研究揭示了ZBP-89调节Bak蛋白表达和肝癌细胞凋亡的表观遗传机制。同时，进一步揭示ZBP-89联合Pin1经由IκB通路调节HDAC3降低的机制. 本研究为肝癌表观遗传学的治疗提供研究基础和科学依据。 / Background / Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide with a very high mortality. Because the success of the conventional therapies is limited, epigenetic therapy may represent an alternative for HCC management. ZBP-89 is known to induce Bak in HCC. However, it is unclear whether epigenetic mechanisms contribute to ZBP-89-mediated Bak. / Histone acetylase 3 (HDAC3) is realized as a chemotherapy target and a biomarker of recurrence in HCC. HDAC3 is frequently overexpressed in HCC and its inhibition enhances the efficacy of anti-HCC chemotherapy. The pilot data have indicated that ZBP-89 reduced HDAC3 in HCC but the mechanism responsible was unknown. The post-translational modification of proteins functions as a key regulatory factor in cellular physiological procedures, such as ubiquitinoylation degradation. As a biomarker of HCC development and recurrence, it is important to understand how ZBP-89 mediates the reduction of HDAC3. / This study focuses on if ZBP-89 regulates Bak expression through epigenetic mechanisms. It is designed to investigate whether DNA methyltransferases (DNMTs), histone acetylases (HDACs) are involved in regulation of ZBP-89-induced Bak expression. The study also elucidates the mechanism how ZBP-89 reduces the level of HDAC3 protein. / Methods and Results / The levels of Bak and ZBP-89 as shown on western blots were reduced but DNMT1 and HDAC3 were increased in HCC cancer tissues compared to the corresponding non-cancer tissues. Co-immunoprecipitation experiments showed that ZBP-89 bound to HDAC3 and DNMT1 but not other epigenetic enzymes, such as HDAC4, DNMT3a and DNMT3b. To clarify if ZBP-89 affects the activities of HDACs and DNMTs, ZBP-89 was overexpressed in HCC cells. Enzyme activities of HDACs and DNMTs were determined using relevant assay kits. Results showed that overexpressed ZBP-89 inhibited the activities of HDACs and DNMTs. Further experiments indicated that ZBP-89-mediated Bak up-regulation might contribute to maintenance of histone H3 and H4 acetylation through inhibition of HDACs activity. In another set of experiments, we also found an increased Bak expression in HCC cells when the cells were treated with HDAC inhibitors (HDACi) VPA and TSA. HDAC3 siRNA also increased Bak expression. / Both knockdown of DNMT1 expression and administration of DNMTs inhibitors (zebularine) induced Bak expression. Chromatin immunoprecipitation (ChIP) showed that ZBP-89 bound to Bak promoter at the region from -3188bp to -3183bp and from -275 to -49. As ZBP-89 inhibits DNMT activity, it is essential to know whether its inhibition affectes DNA CpG methylation status and methyl-CpG binding protein (MeCP) binding. The results showed that ZBP-89 overexpression inhibited MeCP2 binding to genomic DNA. The finding indicated that decreased MeCP2 binding to DNA might be due to decreased methyl-CpG number in Bak promoter, suggesting that ZBP-89 might affect CpG island methylation status. Therefore, the bisulfite modified DNA sequencing method was used to clarify if Bak promoter CpG island methylation status was altered after ZBP-89 overexpression. Results revealed that ZBP-89 overexpression could demethylate the CpG islands in Bak promoter. / ZBP-89 overexpression dose-dependently reduced the expression of HDAC3 at protein level but not at mRNA level. Co-immunoprecipitation and western blot methods were used to analyze Peptidyl-prolyl cis/trans isomerase 1 (Pin1) and HDAC3, phospho-I kappa B (pIκB), and the result revealed that HDAC3 could bound with either Pin1 or pIκB to promote the reduced expression of HDAC3. / Constructed mU6-siPin1 vector was used to knockdown Pin1 expression in HCC cells. We found that knockdown of Pin1 expression blocked ZBP-89-mediated HDAC3 reduction. Experiments performed in Pin1 allele-knockdown JB6 C141 Pin1⁻/⁻ and Pin1⁺/⁺ cells showed that the reduction of HDAC3 by ZBP-89 was greater in Pin1⁺/⁺ cells than in Pin1⁻/⁻ cells, confirming the role of Pin1 in ZBP-89-mediated HDAC3 reduction. Furthermore, the ZBP-89-mediated HDAC3 reduction was suppressed by CAY10576, an IκB kinase (IKK) activation inhibitor but not by SN50, a p65/p50 translocation inhibitor, suggesting that HDAC reduction may depend on IκB kinase rather than NF-κB activity. / HCC xenograft mouse model was used to support the involvement of epigenetic mechanism in ZBP-89-induced Bak expression and its therapeutic effects against HCC. Results showed that ZBP-89 as well as HDAC inhibitor valproic acid (VPA) or/and DNMT inhibitor zebularine stimulated Bak expression and induced apoptosis of tumor cells in an HCC xenograft mouse model, arresting tumor growth. In HCC xenografe model, treatment by injection of Ad-ZBP-89 viral expression vector mediated ZBP-89 expression decreased HDAC3 expression, but not HDAC4. / Conclusions / In conclusion, the study demonstrates a novel mechanism through which ZBP-89 mediates an epigenetic pathway to promote Bak expression, and induce apoptosis in HCC cells. It also reveals the mechanism of HDAC3 reduction by ZBP-89 is dependent on IκB, which requires the presence of Pin1. This pathway may help develop future epigenetic therapy against HCC. / 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. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Ye, Caiguo. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 123-140). / Abstracts also in Chinese. / Abstract --- p.i / 摘要 --- p.v / Publications --- p.viii / Acknowledgements --- p.ix / Abbreviations --- p.xi / List of Tables --- p.xiii / List of figures --- p.xiv / Chapter Chapter One: --- General Introduction --- p.1 / Chapter 1.1 --- Background --- p.2 / Chapter 1.2 --- The complexity of HDAC family and functions --- p.3 / Chapter 1.2.1 --- HDAC family --- p.4 / Chapter 1.2.2 --- Multifunction of HDACs --- p.6 / Chapter 1.3 --- HDACs and apoptosis --- p.6 / Chapter 1.3.1 --- HDAC regulates apoptotic-related gene expression --- p.9 / Chapter 1.3.2 --- HDACs regulate apoptosis through protein complexes --- p.18 / Chapter 1.3.3 --- HDACs mediates non-histone deacetylation and apoptosis --- p.21 / Chapter 1.3.4 --- HDACs degradation deficiency and apoptosis --- p.24 / Chapter 1.4 --- DNMTs and epigenetic modification --- p.25 / Chapter 1.4.1 --- DNMT family --- p.25 / Chapter 1.4.2 --- CpG islands methylation and HCC --- p.26 / Chapter 1.5 --- Perspectives --- p.28 / Chapter Chapter Two: --- ZBP-89 up-regulates Bak expression through inhibition the activity of HDACs and DNMTs --- p.30 / Chapter 2.1 --- Introduction --- p.31 / Chapter 2.2 --- Materials and Methods --- p.33 / Chapter 2.2.1 --- Hepatocellular carcinoma patient samples and cell lines --- p.33 / Chapter 2.2.2 --- Chemicals and reagents --- p.34 / Chapter 2.2.3 --- Cell proliferation --- p.34 / Chapter 2.2.4 --- Adenovirus infection of cells --- p.35 / Chapter 2.2.5 --- Apoptosis detection --- p.36 / Chapter 2.2.6 --- Transfection of siRNA and plasmid --- p.36 / Chapter 2.2.7 --- Co-immunoprecipitation (co-IP) --- p.37 / Chapter 2.2.8 --- Western blotting --- p.37 / Chapter 2.2.9 --- Immunohistochemistry and Immunofluorescence --- p.38 / Chapter 2.2.10 --- Chromatin immunoprecipitation --- p.38 / Chapter 2.2.11 --- Sodium bisulfite modified sequencing of Bak promoter --- p.40 / Chapter 2.2.12 --- Histone deacetylase activity assay --- p.41 / Chapter 2.2.13 --- DNA methyltransferases enzyme activity --- p.42 / Chapter 2.2.14 --- Xenograft animal model --- p.43 / Chapter 2.2.15 --- Statistical analysis --- p.43 / Chapter 2.3 --- Results --- p.45 / Chapter 2.3.1 --- ZBP-89 interacts with DNMT1 and HDAC3 --- p.45 / Chapter 2.3.2 --- DNA methyltransferase-1 and histone deacetylase 3 are overexpressed in cancer tissues --- p.48 / Chapter 2.3.3 --- Inhibition of HDACs and DNMTs induces Bak expression and apoptosis --- p.58 / Chapter 2.3.4 --- Adenovirus mediated ZBP-89 expression inhibits HDACs activity --- p.65 / Chapter 2.3.5 --- ZBP-89 suppresses DNMTs activity --- p.67 / Chapter 2.3.6 --- Overexpressed ZBP-89 demethylates methyl-CpG islands --- p.69 / Chapter 2.3.7 --- Downregulation of HDAC3 and DNMT1 enhances Bak expression --- p.74 / Chapter 2.3.8 --- Xenograft nude mouse model reveals that Ad-ZBP-89 adenovirus diminishes tumor volume and induces Bak expression and apoptosis --- p.75 / Chapter 2.4 --- Discussion --- p.81 / Chapter Chapter Three: --- ZBP-89 targets IkappaB to reduce HDAC3 via a Pin1-dependent pathway --- p.86 / Chapter 3.1 --- Introduction --- p.87 / Chapter 3.2 --- Materials and Methods --- p.89 / Chapter 3.2.1 --- Cell lines, chemicals and reagents --- p.89 / Chapter 3.2.2 --- Transfection of siRNA plasmid --- p.89 / Chapter 3.2.3 --- Plasmid extraction by mini-prep --- p.90 / Chapter 3.2.4 --- Co-immunoprecipitation (co-IP) and Western blotting --- p.91 / Chapter 3.2.5 --- Total RNA extraction --- p.92 / Chapter 3.2.6 --- Reverse transcription and real-time PCR --- p.93 / Chapter 3.2.7 --- Immunohistochemistry and Immunofluorescence --- p.94 / Chapter 3.2.8 --- Xenograft animal model --- p.95 / Chapter 3.2.9 --- Statistical analysis --- p.95 / Chapter 3.3 --- Results --- p.97 / Chapter 3.3.1 --- ZBP-89 overexpression diminishes HDAC3 expression but not HDAC4 --- p.97 / Chapter 3.3.2 --- Knockdown of Pin1 blocks ZBP-89-mediated HDAC3 reduction --- p.99 / Chapter 3.3.3 --- ZBP-89 reduces the level of IκB --- p.103 / Chapter 3.3.4 --- IκB degradation inhibitors suppresses ZBP-89-meditaed HDAC3 reduction --- p.105 / Chapter 3.3.5 --- ZBP-89 decreases HDAC3 but increases Bak in xenograft tumor tissues --- p.111 / Chapter 3.4 --- Discussion --- p.115 / Chapter Chapter Four: --- Conclusions and Future Perspectives --- p.119 / Chapter 4.1 --- Summary of results --- p.120 / Chapter 4.2 --- Conclusions --- p.121 / Chapter 4.3 --- Future Perspectives --- p.121 / References --- p.123

Pang, Pei Shin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 96-109). / Abstract also in Chinese. / Acknowledgement --- p.i / Abstract (English) --- p.ii / Abstract (Chinese) --- p.iv / Table of Content --- p.vi / List of Figures --- p.x / List of Tables --- p.xiii / Abbreviations --- p.xiv / Chapter CHAPTER ONE: --- INTRODUCTION --- p.1 / Chapter 1.1 --- Incidence --- p.2 / Chapter 1.2 --- Etiological Factors --- p.7 / Chapter 1.2.1 --- Viral Hepatitis Infection --- p.7 / Chapter 1.2.1.1 --- Hepatitis B Virus --- p.10 / Chapter 1.2.1.2 --- Hepatitis C Virus --- p.11 / Chapter 1.2.2 --- Cirrhosis and Chronic Inflammation --- p.14 / Chapter 1.2.3 --- Dietary Aflatoxin Contamination --- p.16 / Chapter 1.2.4 --- Obesity --- p.16 / Chapter 1.3 --- Genomic Aberrations of HCC --- p.17 / Chapter 1.3.1 --- Chromosomal Imbalances --- p.17 / Chapter 1.3.2 --- Candidate Tumour Suppressor Genes and Oncogenes in HCC --- p.18 / Chapter 1.3.2.1 --- Chr 1q21-q22 gain --- p.18 / Chapter 1.3.2.2 --- Chr 8p21-p23 loss and 8q21-q24 gain --- p.18 / Chapter 1.3.2.3 --- Chr 13ql2-ql4 loss --- p.19 / Chapter 1.3.2.4 --- Chr 17pl3 loss --- p.20 / Chapter 1.3.3 --- Chromosomal Rearrangement --- p.21 / Chapter 1.4 --- Cell Lines as In-vitro Study Models --- p.22 / Chapter 1.5 --- Aim of study --- p.23 / Chapter 1.5.1 --- Objectives --- p.24 / Chapter CHAPTER TWO: --- MATERIALS AND METHODS --- p.25 / Chapter 2.1 --- Materials --- p.26 / Chapter 2.2 --- Cell Lines and Cell Culture --- p.29 / Chapter 2.2.1 --- Cell Lines --- p.29 / Chapter 2.2.2 --- Cell Culture --- p.29 / Chapter 2.3 --- Comparative Genomic Hybridization (CGH) --- p.30 / Chapter 2.4 --- Spectral Karyotyping (SKY) --- p.31 / Chapter 2.5 --- Expression Profiling --- p.34 / Chapter 2.6 --- Functional Investigations --- p.37 / Chapter 2.6.1 --- Growth Kinetics --- p.37 / Chapter 2.6.2 --- Cytotoxic Assay --- p.38 / Chapter CHAPTER THREE: --- RESULTS --- p.39 / Chapter 3.1 --- Molecular Cytogenetic Analysis --- p.40 / Chapter 3.1.1 --- Comparative Genomic Hybridization (CGH) --- p.40 / Chapter 3.1.1.1 --- Introduction --- p.40 / Chapter 3.1.1.2 --- CGH Results --- p.41 / Chapter 3.1.1.3 --- Clustering Analysis of CGH Data --- p.49 / Chapter 3.1.2 --- Spectral Karyotyping (SKY) --- p.51 / Chapter 3.1.2.1 --- Introduction --- p.51 / Chapter 3.1.2.2 --- Ploidy Status --- p.51 / Chapter 3.1.2.3 --- Structural Rearrangements --- p.54 / Chapter 3.1.2.4 --- Chromosomes Susceptible to Further Rearrangements --- p.63 / Chapter 3.1.2.5 --- Maintained Common HCC Translocations --- p.65 / Chapter 3.2 --- Expression Profiling --- p.67 / Chapter 3.2.1 --- Introduction --- p.67 / Chapter 3.2.2 --- Gene Expression Profiling --- p.69 / Chapter 3.2.3 --- The Ontologies of Deregulated Genes --- p.71 / Chapter 3.2.4 --- Maintained Biological Pathways --- p.74 / Chapter 3.3 --- Functional Investigation --- p.76 / Chapter 3.3.1 --- Introduction --- p.76 / Chapter 3.3.2 --- Cell Morphology --- p.76 / Chapter 3.3.3 --- Growth Kinetics --- p.79 / Chapter 3.3.4 --- Cytotoxic Assay --- p.83 / Chapter CHAPTER FOUR: --- DISCUSSION --- p.86 / Chapter 4.1 --- Introduction --- p.87 / Chapter 4.2 --- Molecular Cytogenetic Analysis --- p.87 / Chapter 4.3 --- Expression Profiling --- p.91 / Chapter 4.4 --- Conclusion --- p.94 / REFERENCES --- p.95

Lau, Chi Chiu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 133-142). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Leung, Kin Chung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 148-164). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

by Shuk-kei Lau. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 141-148). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Table of Contents --- p.ii / Abstract --- p.vi / 論文摘要 --- p.viii / Abbreviations --- p.ix / List of Figures --- p.x / List of Tables --- p.xii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- HBV and its role in hepatocarcinogenesis --- p.3 / Chapter 1.2.1 --- Current situation of HBV infection and the HCC incidencein the world --- p.3 / Chapter 1.2.2 --- Current situation of HBV infection and the HCC incidencein Hong Kong --- p.4 / Chapter 1.2.3 --- Genetic organization of HBV --- p.4 / Chapter 1.2.4 --- Principle of hepatocarcinogenesis induced by HBV --- p.5 / Chapter 1.2.4.1 --- Role of chronic hepatitis in hepatocarcinogenesis --- p.5 / Chapter 1.2.4.2 --- Role of HBV in hepatocarcinogenesis --- p.6 / Chapter 1.2.5 --- Current screening tests for HCC --- p.7 / Chapter 1.2.6 --- Current therapies for HCC --- p.9 / Chapter 1.3 --- Aim of the present study --- p.13 / Chapter 1.4 --- "Combining Expressed Sequence Tag (EST), Suppression Subtractive Hybridization and cDNA microarray for rapid differentially by expressed genes screening" --- p.14 / Chapter 1.4.1 --- Expressed Sequence Tag (EST) --- p.14 / Chapter 1.4.2 --- cDNA subtraction --- p.15 / Chapter 1.4.3 --- cDNA microarray --- p.16 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- PCR-select cDNA subtraction --- p.17 / Chapter 2.1.1 --- Amplification of subtracted cDNA clones by PCR --- p.17 / Chapter 2.1.2 --- Cycle sequencing of subtracted cDNA clones --- p.18 / Chapter 2.1.3 --- Sequence analysis using BLAST server and Stanford Online Universal Resource for Clones and ESTs (SOURCE) --- p.19 / Chapter 2.2 --- cDNA microarray analysis --- p.20 / Chapter 2.2.1 --- Array fabrication --- p.20 / Chapter 2.2.1.1 --- Amplification of cDNA clones by PCR --- p.20 / Chapter 2.2.1.2 --- Purification of PCR products --- p.21 / Chapter 2.2.1.3 --- Cycle sequencing for clones checking --- p.22 / Chapter 2.2.2 --- Microarray printing --- p.22 / Chapter 2.2.2.1 --- Preparation of cDNA target --- p.22 / Chapter 2.2.2.2 --- Arraying --- p.22 / Chapter 2.2.3 --- Screening of differentially expressed genes in hepatocellular carcinoma and its surrounding normal counterpart by cDNA microarray --- p.23 / Chapter 2.2.3.1 --- Extraction of RNA --- p.23 / Chapter 2.2.3.2 --- RNA labeling --- p.24 / Chapter 2.2.3.3 --- Microarray hybridization --- p.26 / Chapter 2.2.3.4 --- Collection of data --- p.27 / Chapter 2.2.3.5 --- Data normalization and analysis --- p.28 / Chapter 2.3 --- Molecular cloning and characterization of a novel cDNA clone differentially expressed in HCC --- p.30 / Chapter 2.3.1 --- Tissue distribution of T2L522 gene --- p.30 / Chapter 2.3.1.1 --- Northern hybridization --- p.30 / Chapter 2.3.1.2 --- Reverse-transcriptase polymerase chain reaction (RT-PCR) --- p.33 / Chapter 2.3.2 --- Expression level of T2L522 in HCC and its surrounding normal counterpart --- p.33 / Chapter 2.3.3 --- Identification of interacting partner of T2L522 using yeast two-hybrid assay --- p.35 / Chapter 2.3.3.1 --- "Cloning of T2L522 gene into the yeast two-hybrid DNA-BD vector, pGBKT7" --- p.35 / Chapter 2.3.3.2 --- Transformation of yeast competent cells --- p.39 / Chapter 2.3.3.3 --- Mating of T2L522-BD with pretransformed human liver cDNA library --- p.40 / Chapter 2.3.3.4 --- Colony lift p-galactosidase filter assay --- p.42 / Chapter 2.3.4 --- Subcellular localization of T2L522 gene by tagging with green fluorescence protein (GFP) --- p.43 / Chapter 2.3.4.1 --- "Cloning of T2L522 gene into the eukaryotic GFP expression vector, pEGFP-Cl" --- p.43 / Chapter 2.3.4.2 --- Transfection of pEGFP-T2L522 into HepG2 cell --- p.43 / Chapter Chapter 3 --- Results / Chapter 3.1 --- PCR-select cDNA subtraction --- p.45 / Chapter 3.1.1 --- The sequencing results of subtracted-HCC cDNA clones --- p.45 / Chapter 3.1.2 --- Categorization of ESTs sequenced from subtracted-HCC library --- p.45 / Chapter 3.2 --- Microarray analysis --- p.49 / Chapter 3.2.1 --- Array fabrication --- p.49 / Chapter 3.2.1.1 --- Amplification of cDNA microarray targets --- p.49 / Chapter 3.2.2 --- Microarray printing --- p.52 / Chapter 3.2.3 --- Microarray analysis of differentially expressed genesin hepatocellular carcinoma and its surrounding normal counterpart --- p.55 / Chapter 3.2.4 --- Data collection --- p.57 / Chapter 3.2.5 --- Image processing: spots finding and quantitation --- p.61 / Chapter 3.2.6 --- Data normalization and analysis --- p.61 / Chapter 3.3 --- Molecular cloning and characterization of a novel cDNA clone differentially expressed in HCC --- p.73 / Chapter 3.3.1 --- Tissue distribution of T2L522 --- p.77 / Chapter 3.3.1.1 --- Northern hybridization --- p.77 / Chapter 3.3.1.2 --- Reverse-transcriptase polymerase chain reaction (RT-PCR) --- p.79 / Chapter 3.3.2 --- Expression level of T2L522 in hepatocellular carcinoma and its surrounding normal counterpart --- p.81 / Chapter 3.3.3 --- Identification of interacting partner of T2L522 using yeast two-hybrid assay --- p.85 / Chapter 3.3.4 --- Subcellular localization of GFP tagged T2L522 --- p.87 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- EST analysis on subtracted-HCC cDNA library --- p.89 / Chapter 4.2 --- cDNA microarray analysis --- p.92 / Chapter 4.2.1 --- Generation of reliable data using cDNA microarray --- p.92 / Chapter 4.2.1.1 --- Reproducibility of signal and normalized ratio --- p.92 / Chapter 4.2.2 --- Comparison of data between multiple slides --- p.96 / Chapter 4.2.2.1 --- Assession of data quality and statistical significance --- p.96 / Chapter 4.2.2.2 --- Interpretation of gene expression data from single and multiple hybridizarion --- p.97 / Chapter 4.3 --- Candidate genes differentially expressed in HCC and its surrounding normal counterpart --- p.99 / Chapter 4.3.1 --- Protein up-regulated in HCC --- p.99 / Chapter 4.3.1.1 --- Extracellular matrix protein --- p.99 / Chapter 4.3.1.2 --- Protein involved in other metabolism --- p.100 / Chapter 4.3.1.3 --- Protein involved in transcription and translation --- p.100 / Chapter 4.3.2 --- Protein down-regulated in HCC --- p.101 / Chapter 4.3.2.1 --- Membrane associated protein --- p.101 / Chapter 4.3.2.2 --- Protein involved in other metabolism --- p.102 / Chapter 4.3.2.2 --- Secretory protein --- p.104 / Chapter 4.3.3 --- Novel protein differentially expressed in HCC --- p.107 / Chapter 4.4 --- "TBC1 domain containing protein, T2L522" --- p.108 / Chapter 4.4.1 --- Possible involvement of T2L522 gene in HCC --- p.109 / Chapter 4.4.2 --- Tissue distribution and expression pattern of T2L522 --- p.110 / Chapter 4.4.3 --- Potential interacting partner of T2L522 --- p.110 / Chapter 4.4.4 --- Subcellular localization of T2L522 --- p.112 / Chapter 4.5 --- Summary --- p.113 / Appendix --- p.114 / References --- p.141

BTBD7 is a newly identified candidate gene for HCC using a high-throughput cDNA/EST microassay. This gene encodes for a protein of 410 amino acid residues. This protein was previously named as the function unknown protein 1 (FUP1) because the biological function of this protein was unknown at that time. Bioinformatics analysis revealed that this protein contains two bric-a-brac, tramtrack, broad-complex (BTB) domains located at amino acid positions 143 to 230 and 274 to 342. In order to reflect its structure and functions, and to be consistent with the GeneBank database (Accession No. NM_018167), we rename it as BTBD7 (BTB domain containing 7). / In conclusion, our study demonstrated that BTBD7 is a novel oncogene, which is associated with hepatocellular carcinoma and is essential for the inhibition of cell growth and tumorigenesis. To our knowledge, BTBD7 is the first identified regulator of p16INK4A through inhibiting the promoter activity of p16INK4A. BTBD7 may thus serve as a new tumor marker or as a potential target of treating hepatocellular carcinoma. / In previous studies, the expression of BTBD7 was shown to be tissue-specific as demonstrated by Northern blot. Furthermore, we collected 18-paired HCC samples to further reveal the correlation of BTBD7 gene expression profiles with tumorigenesis. Our data showed that BTBD7 was significantly elevated in 44.4% of the HCC samples. Compared with immortalized hepatocyte cell lines MIHA or LO2, both mRNA level and protein level of BTBD7 were also elevated in the hepatoma cell lines HepG2, BEL7404, Hep3B and Huh7. This gave a due that the expression of BTBD7 may be correlated with carcinogenesis of liver cells. / In the present study, the function of BTBD7 was investigated. We used RNAi approach to silence BTBD7. Compared with the control, siBTBD7 induced cell cycle arrest at G1 phase and later caused obvious cell death. The cell death was further demonstrated to be apoptosis through activation of caspase 3. Furthermore, we carried out candidate gene search using knockdown of BTBD7. The mRNA level of tumor suppresser p16INK4A was upregulated and hTERT was downregulated in BTBD7 knocked down cells. The other key genes involved in cell growth, cell cycle control, cell death and survival (c-myc, c-fos, c-jun, p21CIP1, p27KIP1, p53, Survivin, E2F, NF-kappaB, Bax, p14ARF, p16INK4A and hTERT) did not respond to the reduced BTBD7 levels. On the other hand, double knockdown of p16INK4A and BTBD7 markedly reduced the effects of cell cycle arrest and the death ratio caused by dysfunction of BTBD7 or overexpression of p16INK4A, suggesting that p16 INK4A is a downstream target of BTBD7. We further adopted a dominant negative approach to confirm these results. / Liu, Zheng. / Advisers: C. H. K. Cheng; Mingliang He. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3449. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 120-161). / 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.

"Epigenetic" refers to a heritable change in the gene expression pattern that is not mediated by any alterations in the primary nucleotide sequence of a gene in the genome. This change involves methylation of DNA in the gene promoter regions, modification of histone residues and chromatin remodeling. Among them, methylation of DNA promoter region is an essential step in epigenetic gene silencing and is known to be closely related to carcinogenesis and cancer progression. / Our preliminary study on effect of treatments of some potential anti-cancer drug candidates, namely Pheophorbide A (Pa), Pa combining with photodynamic therapy, Polyphyllin D (designated as HK-18), and its derivative designated as HK-27 on human breast cancer cell lines MCF-7 and MDA-MB-231 showed that the promoter methylation of CDH1 was decreased in response to treatments of Pa, HK-18, and HK-27 in MDA-MB-231 cells. / The aim of this study was to explore whether any methylation of DNA promoters mechanism is involved in drug resistance of a doxorubicin-induced human multidrug resistant hepatocellular carcinoma sub-linage R-HepG2 which was established from the doxorubicin sensitive HepG2 cell line in our laboratory. In this project, it was observed that the DNA promoter methylations of ESR1, Rassf2A, CDH1 and MDR1 in R-HepG2 were higher than those in HepG2 cells respectively by methylation specific polymerase chain reaction method. Bisulfite sequencing showed that the total 32 CpGs of CDH1 promoter region in R-HepG2 cells were hypermethylated while they were hypomethylated in HepG2 cells. CDH1 is the encoding gene of E-cadherin. The promoter hypermethylation induced CDH1 silencing in R-HepG2 cells was confirmed by reverse transcription polymerase chain reaction and Western blotting that CDH1 transcription and E-cadherin expression were maintained in HepG2 cells but both were lost in R-HepG2 cells. RT-PCR of 10 multidrug resistant related genes revealed that transcription of MDR1 was obviously increased in R-HepG2 cells, transcription of MRP1 and MRP5 were slightly increased in R-HepG2 cells, transcription of MRP6 and BCRP were slightly decreased in R-HepG2 cells comparing to those in the parental HepG2 cells. This result suggests that up-regulation of P-glycoprotein expression which is the protein product of MDR1 may be one of the major causes of multidrug resistance in R-HepG2 cells. Transient transfection of CDH1 cDNA increased the CDH1 transcription and E-cadherin expression in R-HepG2 cells. I also found that the CDH1 transfected R-HepG2-CDH1 cells showed increased amount of doxorubicin uptake, increased apoptotic population of cells exposed to doxorubicin, suppressed cell migration, and decreased P-glycoprotein expression comparing to those in R-HepG2 cells. It was also found that the transcription levels of SNAI2, TWIST1, ASNA1 and FYN were obviously higher in R-HepG2 cells than those in HepG2 cells. The transcription of FYN and TWIST1 were obviously decreased in CDH1 cDNA transfected R-HepG2-CDH1 cells which displayed a negative correlation with the transcription level of CDH1 and these results imply a suppressive role of CDH1 in regulating these genes which were involved in cancer metastasis and multidrug resistance. / Jiang, Lei. / Adviser: Kwok-Pui, Fang. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 144-171). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Cheung Hoi Hung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 151-166). / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.I / ABSTRACT --- p.II / ABBREVIATIONS --- p.IV / LIST OF FIGURES --- p.VII / LIST OF TABLES --- p.IX / CONTENTS --- p.X / Chapter CHAPTER ONE: --- GENERAL INTRODUCTION / Chapter 1.1 --- Non-coding RNAs in transcriptional output --- p.2 / Chapter 1.2 --- Diverse functions of non-coding RNAs --- p.5 / Chapter 1.3 --- HI9: imprinted non-coding RNA --- p.6 / Chapter 1.4 --- Objective --- p.7 / Chapter CHAPTER TWO: --- The ROLE OF H19 RNA IN MDR1 EXPRESSION OF HUMAN HEPATOCELLULAR CARCINOMA HepG2 CELLS / Chapter 2.1 --- Introduction / Chapter 2.1.1 --- H19-Igf2 locus as a model for genomic imprinting --- p.10 / Chapter 2.1.2 --- HI9 as a non-protein coding regulatory RNA --- p.12 / Chapter 2.1.3 --- Controversial roles of H19 RNA --- p.13 / Chapter 2.1.4 --- Novel role of H19 RNA in drug resistance --- p.15 / Chapter 2.2 --- Materials and methods / Chapter 2.2.1 --- Materials --- p.17 / Chapter 2.2.2 --- Methods / Chapter 2.2.2.1 --- Cell culture --- p.19 / Chapter 2.2.2.2 --- Plasmid construction and stable cell transfection --- p.19 / Chapter 2.2.2.3 --- Transient gene transfection --- p.20 / Chapter 2.2.2.4 --- RNA isolation and RT-PCR --- p.21 / Chapter 2.2.2.5 --- MTT drug sensitivity assay --- p.22 / Chapter 2.2.2.6 --- Western blot analysis --- p.22 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Differential expression of H19 RNA in different human cancer cell lines --- p.24 / Chapter 2.3.2 --- R-HepG2 cells over-expressed P-glycoprotein and H19 RNA --- p.24 / Chapter 2.3.3 --- Development of H19-silenced cell lines in HepG2 cells by RNA interference --- p.26 / Chapter 2.3.4 --- Altered drug sensitivity in H19-silenced cells --- p.28 / Chapter 2.3.5 --- Expression of P-glycoprotein in H19-silenced cells --- p.31 / Chapter 2.3.6 --- Overexpression of H19 RNA in HepG2 cells --- p.34 / Chapter 2.3.7 --- Induction of H19 RNA and MDR1 in HepG2 cells --- p.34 / Chapter 2.4 --- Discussion / Chapter 2.4.1 --- H19 regulation of MDR1 associated drug resistance --- p.38 / Chapter 2.4.2 --- The puzzle of riboregulation in drug resistance --- p.40 / Chapter CHAPTER THREE: --- The ROLES OF PTB AND IMP1 IN H19-RELATED MDR1 EXPRESSION OF HUMAN HEPATOCELLULAR CARCINOMA HepG2 CELLS / Chapter 3.1 --- Introduction / Chapter 3.1.1 --- H19 RNA binding proteins --- p.43 / Chapter 3.2 --- Materials and methods / Chapter 3.2.1 --- Materials --- p.46 / Chapter 3.2.2 --- Methods / Chapter 3.2.2.1 --- Cell culture --- p.48 / Chapter 3.2.2.2 --- Plasmid construction and stable cell transfection --- p.48 / Chapter 3.2.2.3 --- RNA extraction and RT-PCR --- p.48 / Chapter 3.2.2.4 --- MTT drug sensitivity assay --- p.48 / Chapter 3.2.2.5 --- Western blot analysis --- p.48 / Chapter 3.2.2.6 --- Real-time PCR analysis of gene expression --- p.49 / Chapter 3.2.2.7 --- DOX efflux assay --- p.49 / Chapter 3.3 --- Results / Chapter 3.3.1 --- PTB knockdown increased P-glycoprotein expression --- p.51 / Chapter 3.3.2 --- IMP1 knockdown decreased MDR1 /P-glycoprotein expression --- p.54 / Chapter 3.3.3 --- Altered drug sensitivity in IMP 1 -knockdown cells --- p.60 / Chapter 3.4 --- Discussion / Chapter 3.4.1 --- Antagonistic effect of PTB and IMP1 on H19/MDR1 expressions --- p.64 / Chapter 3.4.2 --- Complexity of riboregulation --- p.65 / Chapter CHAPTER FOUR: --- IDENTIFICATION OF H19 RNA BINDING PROTEINS FROM HUMAN HEPATOCELLULAR CARCINOMA HepG2 CELLS / Chapter 4.1 --- Introduction / Chapter 4.1.1 --- Overview of RNA-protein interactions --- p.69 / Chapter 4.1.2 --- Methodology in the study of RNA-protein interactions --- p.71 / Chapter 4.1.3 --- Identification of RNA-binding proteins --- p.72 / Chapter 4.2 --- Materials and methods / Chapter 4.2.1 --- Materials --- p.75 / Chapter 4.2.2 --- Methods / Chapter 4.2.2.1 --- Screening of H19 cDNA from human placenta cDNA library --- p.78 / Chapter 4.2.2.2 --- Preparation of nuclear and cytoplasmic extracts from HepG2 cells / Chapter 4.2.2.3 --- In vitro RNA transcription and RNA labeling --- p.80 / Chapter 4.2.2.4 --- RNA electrophoretic mobility shift assay --- p.81 / Chapter 4.2.2.5 --- In vitro UV-crosslinking assay --- p.82 / Chapter 4.2.2.6 --- Preparation of RNA-affinity column and isolation of RNA binding proteins --- p.83 / Chapter 4.2.2.7 --- In-gel digestion and MALDI-TOF mass spectrometry --- p.84 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Screening of H19 cDNA and preparation ofH19 RNA --- p.86 / Chapter 4.3.2 --- Electrophoretic mobility shift analysis of H19 RNA with HepG2 cytoplasmic extract --- p.87 / Chapter 4.3.3 --- UV-crosslinking of H19 RNA with HepG2 nuclear and cytoplasmic extract --- p.90 / Chapter 4.3.4 --- Isolation of H19 RNA binding proteins by RNA-affmity chromatography --- p.94 / Chapter 4.3.5 --- Confirmation of PTB and IMP1 as H19 RNA binding protein --- p.96 / Chapter 4.3.6 --- MALDI-TOF mass spectrometric analysis of isolated H19 RNA binding proteins --- p.96 / Chapter 4.4 --- Discussion / Chapter 4.4.1 --- RNA-protein interactions: an initial step for mechanistic study --- p.99 / Chapter 4.4.2 --- In vitro and in vivo methods for isolation of RNA binding proteins --- p.101 / Chapter 4.4.3 --- Novel role of hnRNP M protein in H19 RNA binding --- p.103 / Chapter CHAPTER FIVE: --- THE ROLE OF PTB IN APOPTOSIS / Chapter 5.1 --- Introduction / Chapter 5.1.1 --- Overview of polypyrimidine tract-binding protein in RNA processing and post-transcriptional gene regulation --- p.106 / Chapter 5.1.2 --- Evidences of polyrimidine-tract binding protein in the regulation of apoptosis --- p.108 / Chapter 5.2 --- Materials and methods / Chapter 5.2.1 --- Materials --- p.111 / Chapter 5.2.2 --- Methods / Chapter 5.2.2.1 --- Cell culture --- p.114 / Chapter 5.2.2.2 --- Stable cell transfection in A431 cells --- p.114 / Chapter 5.2.2.3 --- Western Blot analysis --- p.114 / Chapter 5.2.2.4 --- MTT drug sensitivity assay --- p.114 / Chapter 5.2.2.5 --- DNA fragmentation assay --- p.115 / Chapter 5.2.2.6 --- Flow cytometry analysis of apoptosis --- p.115 / Chapter 5.2.2.7 --- Caspase activity assay --- p.116 / Chapter 5.3 --- Results / Chapter 5.3.1 --- Taxol as an apoptosis inducer in HepG2 cells --- p.117 / Chapter 5.3.2 --- PTB was cleaved during Taxol-induced apoptosis --- p.118 / Chapter 5.3.3 --- PTB knockdown increased Taxol cytotoxicity and apoptosis --- p.118 / Chapter 5.3.4 --- Effect of PTB knockdown on drug sensitivity of cells --- p.121 / Chapter 5.3.5 --- Effect of PTB knockdown on other drug-induced apoptosis --- p.121 / Chapter 5.3.6 --- Effect of PTB knockdown on the basal expressions of genes in apoptosis pathway --- p.126 / Chapter 5.3.7 --- The role of caspase-9 activation in PTB-regulated apoptosis --- p.129 / Chapter 5.3.8 --- The effect of PTB knockdown on pro-caspase-9 expression and Taxol-induced apoptosis in A431 cells --- p.133 / Chapter 5.3.9 --- The role of PTB in the regulation of intrinsic apoptosis pathway --- p.136 / Chapter 5.4 --- Discussion / Chapter 5.4.1 --- The role of PTB in intrinsic apoptosis pathway --- p.138 / Chapter 5.4.2 --- PTB in regulation of pro-caspase-9 expression --- p.139 / Chapter CHAPTER SIX: --- GENERAL DISCUSSION AND CONCLUSION / Chapter 6.1 --- H19 as a potential target in anti-cancer gene therapy --- p.143 / Chapter 6.2 --- Conclusion --- p.144 / Chapter 6.3 --- Unanswered questions and future work --- p.145 / Chapter 6.4 --- A proposed model for H19 pathway --- p.148 / REFERENCES --- p.151