Functional characterization of GEF-H1 in liver tumorigenesis.

Tsang, Chi Keung."November 2011."Thesis (M.Phil.)--Chinese University of Hong Kong, 2012.Includes bibliographical references (leaves 103-116).Abstracts in English and Chinese.Abstract --- p.I摘要 --- p.IIIAcknowledgement --- p.IVTable of content --- p.VList of Figures --- p.VIIIList of Tables --- p.XIAbbreviations --- p.XIIChapter Chapter 1: --- INTRODUCTION --- p.1Chapter 1.1. --- Hepatocellular carcinoma --- p.2Chapter 1.1.1. --- Etiological factors --- p.11Chapter 1.1.1.1. --- Chronic Hepatitis and Liver Cirrhosis --- p.13Chapter 1.1.1.2. --- HBV --- p.13Chapter 1.1.1.3. --- HCV --- p.17Chapter 1.1.1.4. --- Male gender --- p.20Chapter 1.1.1.5. --- Aflatoxin B1 exposure --- p.21Chapter 1.2. --- Genomic abnormalities in HCC --- p.23Chapter 1.3. --- GEF-H1 --- p.24Chapter 1.4. --- RhoA --- p.26Chapter 1.5. --- Epithelial-Mesenchymal Transition (EMT) --- p.29Chapter 1.6. --- Aims of Thesis --- p.31Chapter Chapter 2: --- MATERIALS AND METHODS --- p.32Chapter 2.1. --- Materials --- p.33Chapter 2.1.1. --- Chemicals and Reagents --- p.33Chapter 2.1.2. --- Buffers --- p.35Chapter 2.1.3. --- Cell Culture --- p.37Chapter 2.1.4. --- Nucleic Acids --- p.38Chapter 2.1.5. --- Enzymes --- p.39Chapter 2.1.6. --- Equipments --- p.40Chapter 2.1.7. --- Kits --- p.41Chapter 2.1.8. --- Antibodies --- p.42Chapter 2.1.9. --- Software and Web Resources --- p.43Chapter 2.2. --- Fluorescence In Situ Hybridization (FISH) --- p.44Chapter 2.2.1. --- Probe Preparation --- p.44Chapter 2.2.1.1. --- Human Bacterial Artificial Chromosome (BAC) probe preparation --- p.44Chapter 2.2.1.2. --- Nick translation --- p.44Chapter 2.2.2. --- Hybridization --- p.45Chapter 2.3. --- Genomic DNA extraction --- p.47Chapter 2.4. --- Copy number analysis --- p.48Chapter 2.5. --- Exon Sequencing analysis --- p.49Chapter 2.5.1. --- PCR amplification of GEF-H1 exons --- p.49Chapter 2.5.2. --- Cycle sequencing --- p.49Chapter 2.6. --- Ectopic expression of GEF-H1 in immortalized hepatocyte cell line --- p.52Chapter 2.6.1. --- Construction of GEF-H1 expressing vector --- p.52Chapter 2.6.2. --- Sub-cloning --- p.52Chapter 2.6.3. --- Transfection and clonal selection --- p.53Chapter 2.7. --- Gene Expression Analysis by Quantitative RT-PCR --- p.55Chapter 2.7.1. --- Total RNA extraction --- p.55Chapter 2.7.2. --- qRT-PCR analysis for gene expression --- p.55Chapter 2.8. --- Western blot --- p.58Chapter 2.9. --- Functional Analysis --- p.60Chapter 2.9.1. --- Cell viability (MTT) assay --- p.60Chapter 2.9.2. --- Cell proliferation assays (BrdU-incorporation) --- p.60Chapter 2.9.3. --- Mitomycin C treatment --- p.61Chapter 2.9.4. --- Migration and Invasion assays --- p.63Chapter 2.9.5. --- Wound healing assay --- p.65Chapter 2.9.6. --- Transient knock-down of RhoA --- p.65Chapter 2. --- 10. Immuno-fluorescent imaging --- p.66Chapter 2. --- 11. In vivo tumorigenic study of GEF-H1 by subcutaneous injection --- p.68Chapter 2. --- 12. Statistical analysis --- p.69Chapter Chapter 3: --- RESULTS --- p.70Chapter 3.1. --- Verifying copy number gain of GEF-H1 in high GEF-H1 expressing HCC --- p.71Chapter 3.2. --- Verifying if there is any GEF-H1 exon point mutation in HCC --- p.75Chapter 3.3. --- Functional roles of GEF-H1 in HCC --- p.77Chapter 3.4. --- GEF-Hl-induced functions were RhoA independent --- p.83Chapter 3.5. --- GEF-H1 Induction of Epithelial-mesenchymal transition in HCC --- p.88Chapter 3.6. --- GEF-H1 induced tumorigenicity of MIHA cells --- p.95Chapter Chapter 4: --- DISCUSSIONS --- p.96Chapter 4.1. --- GEF-H1 in HCC and other cancers --- p.97Chapter 4.2. --- GEF-H1 promotes cell motility --- p.98Chapter 4.3. --- GEF-H1 induced tumorigenicity --- p.100Chapter Chapter 5: --- CONCLUSIONS AND PROPOSED FUTURE INVESTIGATIONS --- p.101Chapter Chapter 6: --- REFERENCES --- p.10