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Functional characterization of cancer-associated fibroblasts in the regulation of cancer stem cell-like properties in hepatocellular carcinomaLau, Yuen-ting, 劉婉婷 January 2015 (has links)
abstract / Pathology / Doctoral / Doctor of Philosophy
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Identification and characterization of novel genetic alterations in the progression of hepatocellular carcinomaLiu, Ming, 劉銘 January 2013 (has links)
Hepatocellular carcinoma (HCC) is one of the most frequent human malignancies worldwide with very poor prognosis. It is generally believed that accumulation of irreversible alterations in critical oncogenes and tumor suppressor genes during the long-term inflammation finally leads to the hepatocellular pathogenesis. Although under intensive investigation, the molecular pathogenesis of HCC still remains to be further elucidated.
In this study, we aimed to identify novel genetic alterations critical to the pathogenesis of HCC, especially in hot regions with recurrent chromosomal instability. Amplification of broad regions of 8q is one of the most frequent genetic alterations in HCC, suggesting the existence of oncogenes in addition to MYC at 8q24. By screening the publicly available microarray database and clinical samples, we found frequent amplification and overexpression of Serum and Glucocorticoid Kinase 3 (SGK3) in clinical HCC specimens, and SGK3 genomic activation was significantly associated with poor outcome of HCC patients. Functional assays revealed that SGK3 could increase G1/S cell cycle progression, cell survival, clonogenicity, anchorage-independent growth, and tumor formation in nude mice. We provided evidences that SGK3 could promote HCC growth and survival through inactivating GSK3-β and BAD respectively. We also found that expression of SGK3, which like AKT is activated by PI3K/PDK1, has more significance than overexpression of AKT in predicting poor outcome of HCC patients. Our findings suggested the existence of an AKT-independent SGK3 pathway, which may function in parallel with AKT pathway in the pathogenesis of HCC. In addition to large chromosomal alterations, small changes in nucleotides may also make substantial contributions to carcinogenesis. Recent advances in high-throughput deep sequencing technology have provided a powerful tool to understand the whole cancer transcriptome and identify novel genetic alterations related to cancer progression. In this study, we identified a high proportion of allele imbalance in genes related to cellular stress response by sequencing the whole transcriptome of 3 paired HCC tissues. A novel nucleotide variation which resulted in a R438H amino acid change was identified in the coding region of the gene Oxidative Stress Induced Growth Inhibitor 1 (OSGIN1), and the variant 438H form of OSGIN1 was found to be specifically retained in the tumor tissues in a cohort of HCC patients. OSGIN1 was found to be closely associated with chemotherapeutic reagents and exhibited strong tumor suppressive function in HCC by directly inducing cell apoptosis. The wild type OSGIN1 was found to have stronger tumor suppressive function than the variant allele, and this might be due to their different ability to localize to mitochondria. The significantly decreased basal apoptotic index in HCC patients carrying OSGIN1 variant allele and their poor prognosis further suggested that the specific retention of 438H OSGIN1 might be important in HCC progression.
In summary, we found a frequently amplified oncogenic SGK3 signaling pathway, as well as the allele-specific imbalance of tumor suppressive OSGIN1 in the pathogenesis of HCC. Further characterization of their mechanisms in hepatocarcinogenesis may help provide novel prognostic biomarkers and therapeutic targets in HCC treatment. / published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy
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MicroRNA profiling of human hepatocytes induced by HBx in hepatocarcinogenesis.January 2009 (has links)
Yip, Wing Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 100-119). / Abstract also in Chinese. / Abstract (English version) --- p.i / Abstract (Chinese version) --- p.iii / Acknowledgments --- p.v / Table of Contents --- p.vii / List of Tables --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xiii / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Hepatocellular Carcinoma --- p.1 / Chapter 1.1.1 --- Epidermiology --- p.1 / Chapter 1.1.2 --- Etiology --- p.1 / Chapter 1.2 --- Hepatitis B Virus --- p.3 / Chapter 1.2.1 --- The Epidermiology of Hepatitis B Virus Infection --- p.3 / Chapter 1.2.2 --- The Morphology and Genome of Hepatitis B Virus --- p.4 / Chapter 1.2.3 --- HBV Genotypes and Their Significance --- p.8 / Chapter 1.3 --- Hepatitis B Virus X Protein --- p.9 / Chapter 1.3.1 --- HBx Alters Various Signal Transduction Pathways --- p.10 / Chapter 1.3.2 --- HBx Interacts with Various Transcription Factors and Co-activators --- p.12 / Chapter 1.3.3 --- HBx Induces Epigenetic Alterations --- p.14 / Chapter 1.3.4 --- Identification of COOH-terminal Truncated HBx in Liver Tumors --- p.15 / Chapter 1.4 --- MicroRNAs --- p.17 / Chapter 1.4.1 --- Transcriptional Regulation and Biogenesis of MicroRNAs --- p.18 / Chapter 1.4.2 --- MicroRNAs and Cancer --- p.21 / Chapter 1.4.3 --- MicroRNAs and HCC --- p.25 / Chapter 1.5 --- Hypothesis and Aims of the Study --- p.29 / Chapter CHAPTER 2 --- MATERIALS and METHODS --- p.30 / Chapter 2.1 --- Patients --- p.30 / Chapter 2.2 --- Cell Lines --- p.30 / Chapter 2.3 --- Cloning of Various HBx Constructs --- p.32 / Chapter 2.3.1 --- PCR Amplification of HBx Fragments --- p.32 / Chapter 2.3.2 --- Cloning of HBx Fragments into TA-vectos --- p.33 / Chapter 2.3.3 --- Heat Shock Transformation --- p.33 / Chapter 2.3.4 --- Sub-cloning of HBx Fragments into Lentiviral Vectors --- p.34 / Chapter 2.4 --- Generation of Lentivirus --- p.37 / Chapter 2.4.1 --- Lentivirus Infection --- p.37 / Chapter 2.5 --- RNA Extraction --- p.38 / Chapter 2.6 --- Western Blot Analysis --- p.39 / Chapter 2.7 --- MiRNA Microarray --- p.40 / Chapter 2.7.1 --- Cyanine3-pCp Labeling of RNA Samples --- p.40 / Chapter 2.7.2 --- Sample Hybridization --- p.41 / Chapter 2.7.3 --- Microarray Wash --- p.41 / Chapter 2.7.4 --- Array Slide Scanning and Processing --- p.41 / Chapter 2.8 --- Detection of HBx Gene Deletion by PCR --- p.43 / Chapter 2.9 --- Immunohistochemistry --- p.44 / Chapter 2.10 --- Quantitative Real-time PCR --- p.45 / Chapter 2.11 --- Proliferation Assay --- p.47 / Chapter 2.12 --- Cell Cycle Analysis --- p.48 / Chapter 2.13 --- Annexin V Apoptosis Assay --- p.49 / Chapter 2.14 --- Colony Formation Assay --- p.50 / Chapter 2.15 --- Statistical Analysis --- p.51 / Chapter CHAPTER 3 --- RESULTS --- p.52 / Chapter 3.1 --- Detection of Full-length and COOH-terminal Truncated HBx in HCC Tissues --- p.52 / Chapter 3.2 --- Confirmation of HBx Expression in HCC Tissues --- p.55 / Chapter 3.3 --- Comparison of HBx from Different HBV Genotypes for Study --- p.61 / Chapter 3.4 --- Functional Characterization of COOH-tterminal Truncated HBx --- p.64 / Chapter 3.4.1 --- Selection of COOH-terminal Truncated HBx --- p.64 / Chapter 3.4.2 --- Generation of Various HBx-expressing Hepatocyte Cell Lines --- p.66 / Chapter 3.4.3 --- Effect of Full-length and COOH-terminal Truncated HBx on Cell Proliferation --- p.69 / Chapter 3.4.4 --- Effect of Full-length and COOH-terminal Truncated HBx Cell Cycle --- p.34 / Chapter 3.4.5 --- Effect of Full-length and COOH-terminal Truncated HBx on Apoptosis --- p.45 / Chapter 3.5 --- MicroRNA Profiling of Various HBx-expressing Hepatocyte Cell Lines --- p.76 / Chapter 3.5.1 --- Identification of Deregulated MicroRNAs by Microarray --- p.76 / Chapter 3.5.2 --- Validation of Deregulated MicroRNAs by Real-time PCR Analysis --- p.80 / Chapter 3.5.3 --- Confirmation of Deregulated MiRNAs in HCC and Adjacent Non-tumor Tissues --- p.84 / Chapter 3.5.4 --- Potential Downstream Targets of the HBx-deregulated MiRNAs --- p.87 / Chapter CHAPTER 4 --- DISCUSSION --- p.91 / Chapter 4.1 --- The Impact of COOH-terminal Truncated HBx in HCC --- p.91 / Chapter 4.2 --- The Biological Significance of COOH-terminal Truncated HBx Induced MiRNAs --- p.94 / Chapter 4.3 --- Limitations of the Present Study --- p.97 / Chapter 4.4 --- Future Studies --- p.98 / Chapter CHAPTER 5 --- CONCLUSION --- p.99 / REFERENCES --- p.100
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Hypoxia acts as an enhancer for the cleavage of BID in HBx-transfected liver cells treated with doxorubicin.January 2009 (has links)
Chau, Kin Fan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 106-119). / Abstract also in Chinese. / Abstract --- p.II / 摘要 --- p.VI / Acknowledgements --- p.IX / List of figures --- p.X / List of Abbreviations --- p.XII / Table of Contents --- p.XV / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Incidence and etiology of hepatocellular carcinoma (HCC) --- p.1 / Chapter 1.2 --- Structure of Hepatitis B Virus (HBV) --- p.2 / Chapter 1.3 --- Hepatitis B X protein (HBx) and HCC --- p.5 / Chapter 1.4 --- HBx and Apoptosis --- p.8 / Chapter 1.5 --- The role of Bcl-2 family in apoptosis and cell survival --- p.10 / Chapter 1.6 --- "Bid, the BH3-domain only protein" --- p.14 / Chapter 1.7 --- Dual Functions of Bid --- p.16 / Chapter 1.8 --- The relationship between Bid and HBx --- p.19 / Chapter 1.9 --- Hypoxia and HCC --- p.21 / Chapter 1.10 --- Hypoxia and HBx --- p.25 / Chapter 1.11 --- Hypoxia and Bid --- p.28 / Chapter 1.12 --- Aim of study --- p.29 / Chapter Chapter 2: --- Methods and materials / Chapter 2.1 --- Confirmation of the culture of the stable cell lines --- p.30 / Chapter 2.2 --- Doxorubicin treatment to the cell lines --- p.34 / Chapter 2.3 --- Culture of the cell lines under hypoxic conditions --- p.35 / Chapter 2.4 --- Protein sample preparations --- p.37 / Chapter 2.5 --- Determination of protein samples --- p.38 / Chapter 2.6 --- Sodium dodecyl sulfate 226}0ؤ polyacrylamide gel electrophoresis (SDS- PAGE) --- p.39 / Chapter 2.7 --- Transfer of protein to nitrocellulose membranes --- p.39 / Chapter 2.8 --- Western blot analysis of proteins --- p.41 / Chapter 2.8.1. --- Antibodies --- p.41 / Chapter 2.8.2. --- Determination of expression profiles of desired proteins by immunoblotting --- p.45 / Chapter 2.9 --- "Measurement of cell viability by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay" --- p.46 / Chapter 2.10 --- Determination of cell proliferation by BrdU proliferation assay --- p.47 / Chapter 2.11 --- Detection of apoptosis of the cell lines by TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling) --- p.50 / Chapter 2.12 --- Determination of the involvement of p38 MAPK in the generation of truncated Bid by p38 MAPK inhibitor SB203580 --- p.52 / Chapter Chapter 3: --- Results / Chapter 3.1 --- Confirmation of plasmids and the stable cell lines --- p.53 / Chapter 3.2 --- Morphology and the basic parameters of the cells with full-length HBx or mutant HBx --- p.53 / Chapter 3.3 --- Cell viability under doxorubicin treatment with or without hypoxia --- p.59 / Chapter 3.4 --- Determination of cell proliferation under stress --- p.70 / Chapter 3.5 --- Expression profiles of various proteins in the stable cell lines under doxorubicin treatment with or without hypoxia --- p.74 / Chapter 3.5.1. --- Verification of hypoxia --- p.74 / Chapter 3.5.2. --- Pro-apoptotic proteins --- p.74 / Chapter 3.5.3. --- Anti-apoptotic proteins --- p.74 / Chapter 3.6 --- Determination of apoptosis of various cell lines under stress --- p.82 / Chapter 3.7 --- "p38 MAPK, but not Akt, was activated by doxorubicin" --- p.87 / Chapter 3.8 --- The p38 MAPK inhibitor SB203580 could attenuate the cleavage of Bid --- p.89 / Chapter Chapter 4: --- Discussion --- p.92 / Chapter Chapter 5: --- Conclusion and future prospective --- p.103 / Chapter Chapter 6: --- References --- p.106
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