Tse, Ho Man. / Thesis (M.Phil)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 166-173). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 撮要 --- p.v / Table of Content --- p.vi / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Basis of Prostate Cancer --- p.1 / Chapter 1.1.1 --- Epidemiology and Risk Factors of Prostate Cancer --- p.1 / Chapter 1.1.2 --- Pathology of Prostate Cancer --- p.2 / Chapter 1.1.3 --- Treatment Approaches for Prostate Cancer --- p.4 / Chapter 1.2 --- Sex Hormones and Prostate Cancer --- p.7 / Chapter 1.2.1 --- Prostate Development --- p.7 / Chapter 1.2.2 --- Involvement of Sex Hormones in Prostate Cancer --- p.8 / Chapter 1.2.3 --- Molecular Mechanisms of Sex Hormones --- p.13 / Chapter 1.2.4 --- Hormone Receptor Antagonists --- p.15 / Chapter 1.3 --- Involvement of microRNAs in Cancer --- p.19 / Chapter 1.3.1 --- Basis of microRNAs --- p.19 / Chapter 1.3.2 --- Aberrant microRNA Expressions in Cancers --- p.23 / Chapter 1.3.3 --- Current Understandings on Regulations of micro RN A Expressions --- p.26 / Chapter 1.3.4 --- Regulation of miRNA Expressions by Hormones --- p.29 / Chapter 1.4 --- "Effects of the Anti-estrogen ICI 182,780 on Prostate Cancer Cells" --- p.30 / Chapter 1.4.1 --- "ICI 182,780 Inhibits Cell Growth ofDU145" --- p.30 / Chapter 1.5 --- Objectives of Project --- p.32 / Chapter Chapter 2: --- Materials --- p.34 / Chapter 2.1 --- Bacteria Strain --- p.34 / Chapter 2.2 --- Tissue Culture Media --- p.34 / Chapter 2.3 --- Plasmids --- p.34 / Chapter 2.4 --- Kits and Accessories --- p.35 / Chapter 2.5 --- Reagents and Solutions --- p.36 / Chapter 2.6 --- DNA Oligos --- p.38 / Chapter 2.7 --- Equipments --- p.40 / Chapter Chapter 3: --- Methods --- p.41 / Chapter 3.1 --- Cell Culture Conditions --- p.41 / Chapter 3.2 --- miRNA Expression Profiling of DU145 --- p.41 / Chapter 3.2.1 --- RNA Isolation --- p.41 / Chapter 3.2.2 --- miRNA Microarray Profiling ofDU145 : --- p.42 / Chapter 3.2.2.1 --- Fluorescent Labeling of RNA and Microarray Hybridization --- p.42 / Chapter 3.2.2.2 --- Scanning and Analysis of Signals --- p.46 / Chapter 3.2.3 --- Confirming miR-765 Up-regulation by ICI with qRT-PCR --- p.46 / Chapter 3.2.3.1 --- Assessing ERp Dependency in miR-765 Induction --- p.48 / Chapter 3.2.4 --- Effects of ICI on ARHGEF11 Expression --- p.49 / Chapter 3.2.4.1 --- Reverse Transcription of mRNA --- p.50 / Chapter 3.2.4.2 --- Quantitative Real-Time PCR for Gene mRNA expression --- p.50 / Chapter 3.3 --- Characterizing the Promoter Region of miR-765 --- p.52 / Chapter 3.3.1 --- Cloning of miR-765 Promoter into pGL3-Basic Vector --- p.52 / Chapter 3.3.1.1 --- PCR Amplification of miR-765 Putative Promoter Region --- p.52 / Chapter 3.3.1.2 --- Ligation of the Amplified Regions in pGL3-Basic Vector --- p.55 / Chapter 3.3.1.3 --- Transformation and Screening of pGL3-765 Plasmid --- p.57 / Chapter 3.3.1.4 --- Preparation of pGL3-765 Plasmid DNA --- p.59 / Chapter 3.3.2 --- Preparation of Truncated miR- 765 Promoter Clones --- p.60 / Chapter 3.3.2.1 --- pGL3-765-Trunc#l --- p.61 / Chapter 3.3.2.2 --- pGL3-765-Trunc#2 --- p.62 / Chapter 3.3.2.3 --- pGL3-765-Trunc#3 --- p.62 / Chapter 3.3.3 --- Assessing the miR- 765 Promoter Activities --- p.63 / Chapter 3.3.3.1 --- Optimizing Transfection Conditions --- p.64 / Chapter 3.3.3.2 --- Co-transfection of pGL3-765 and pRL-CMV into DU145 Cells.. --- p.64 / Chapter 3.3.3.3 --- Measuring Luciferase Activities --- p.65 / Chapter 3.3.4 --- Computational Prediction of Transcription Factor Binding Sites on miR-765 Promoter --- p.66 / Chapter 3.4 --- Characterizing the Promoter Region of ARHGEF11.. --- p.67 / Chapter 3.4.1 --- Cloning of ARHGEF11 Promoter into pGL3-Basic Vector (pGL3-ARH) --- p.67 / Chapter 3.4.1.1 --- PCR Amplification of ARHGEF11 Putative Promoter Region --- p.67 / Chapter 3.4.1.2 --- Ligation of the Amplified Regions in pGL3-Basic Vector --- p.68 / Chapter 3.4.1.3 --- Preparation of Plasmid DNA --- p.69 / Chapter 3.4.2 --- Preparation of Truncated ARHGEF11 Promoter Clones --- p.69 / Chapter 3.4.2.1 --- pGL3-ARH-Trunc#l --- p.69 / Chapter 3.4.2.2 --- pGL3-ARH-Trunc#2 --- p.70 / Chapter 3.4.2.3 --- pGL3-ARH-Trunc#3 --- p.71 / Chapter 3.4.3 --- Assessing ARHGEF11 Promoter Activities --- p.72 / Chapter 3.5 --- Identifying Transcription Factor Binding Sites on ARHGEF11 Promoter with EMS A --- p.73 / Chapter 3.5.1 --- Computational Prediction --- p.73 / Chapter 3.5.2 --- Preparation of Biotinylated Probe for use in EMSA --- p.73 / Chapter 3.5.3 --- Preparation of Specific Competitors --- p.74 / Chapter 3.5.4 --- Preparation of DU145 Nuclear and Cytoplasmic Extracts --- p.75 / Chapter 3.5.4.1 --- Preparation of Extracts --- p.75 / Chapter 3.5.4.2 --- Measuring Protein Concentrations --- p.76 / Chapter 3.5.5 --- EMSA Detection of Interaction between Protein and Probe --- p.76 / Chapter 3.6 --- Assessing Biological Significances of miR-765 --- p.78 / Chapter 3.6.1 --- Effects of ICI on DU145 Cells Growth --- p.79 / Chapter 3.6.2 --- Effects of ICI on DU145 Migration Ability --- p.79 / Chapter 3.6.2.1 --- Monolayer Wound Healing Assay --- p.79 / Chapter 3.6.2.2 --- Transwell Migration Assay --- p.80 / Chapter 3.6.3 --- Validating Functionality of Ectopic miR- 765 --- p.81 / Chapter 3.6.3.1 --- miR-765 Recognition Sequence --- p.81 / Chapter 3.6.3.2 --- Preparation of pMIR-765 vector --- p.82 / Chapter 3.6.3.3 --- Ectopic Introduction of miR-765 into DU145 Cells --- p.84 / Chapter 3.6.3.4 --- "Verifying Functionality, of Ectopic miR-765" --- p.84 / Chapter 3.6.4 --- Effects of miR-765 on DU145 Growth --- p.86 / Chapter 3.6.5 --- Effects of miR-765 on DU145 Migration Ability --- p.86 / Chapter 3.7 --- Statistical Analysis --- p.87 / Chapter Chapter 4: --- Results --- p.88 / Chapter 4.1 --- "Identifying ICI 182,780-Regulated miRNA in DU145 Cells" --- p.88 / Chapter 4.1.1 --- miRNA Expression Profiling of DU145 with Microarray --- p.88 / Chapter 4.1.2 --- "Confirming Induction of miR-765 by ICI 182,780 with qRT-PCR" --- p.91 / Chapter 4.1.3 --- "ARHGEF11, Host Gene of miR-765" --- p.95 / Chapter 4.1.4 --- "Induction of ARHGEF 11 by ICI 182,780" --- p.96 / Chapter 4.2 --- Characterization miR-765 Promoter Region --- p.98 / Chapter 4.2.1 --- Cloning of miR- 765 Promoter Region into pGLS-Basic Vector --- p.98 / Chapter 4.2.2 --- Promoter Activity of miR-765 Promoter --- p.100 / Chapter 4.2.3 --- Deletion Mapping of miR- 765 Promoter Region --- p.102 / Chapter 4.2.4 --- Promoter Activities and Inducibitiy of Truncated miR-765 Promoters --- p.103 / Chapter 4.2.5 --- Computational Prediction of Transcription Factor Binding Sites on miR-765 Promoter --- p.105 / Chapter 4.3 --- Characterization of ARHGEF 11 Promoter Region --- p.107 / Chapter 4.3.1 --- Cloning of ARHGEF 11 Promoter --- p.107 / Chapter 4.3.2 --- Promoter Activitiy of ARHGEFll Promoter --- p.109 / Chapter 4.3.3 --- Deletion Mapping of ARHGEFll Promoter --- p.111 / Chapter 4.3.4 --- Promoter Activities and Inducibitiy of Truncated ARHGEF 11 Promoters --- p.113 / Chapter 4.4 --- Identifying Transcription Factor Binding Sites on ARHGEF 11 Promoter --- p.115 / Chapter 4.4.1 --- Computational Prediction of Transcription Factor Binding Sites onARHGEFll Promoter --- p.115 / Chapter 4.4.2 --- Preparation of Probe and Specific Competitors for EMSA --- p.117 / Chapter 4.4.3 --- Interaction between DU145 Nuclear Extract and ARHGEF 11 Promoter Region --- p.119 / Chapter 4.5 --- Biological Significances of miR-765 --- p.122 / Chapter 4.2.1 --- "Effects of ICI 182,780 on DU145 Cell growth" --- p.122 / Chapter 4.2.2 --- "Effects of ICI 182,780 on DU145 Cell Migration" --- p.124 / Chapter 4.2.3 --- Verifying Functionality of Ectopic miR-765 --- p.131 / Chapter 4.2.4 --- Effects of miR-765 on DU145 Cell Growth --- p.133 / Chapter 4.2.5 --- Effects of miR-765 on DU145 Cell Migration --- p.135 / Chapter Chapter 5: --- Discussion --- p.138 / Chapter 5.1 --- "Identifying miR-765 as an Up-regulated miRNA by ICI 182,780" --- p.139 / Chapter 5.1.1 --- "Information about ICI 182,780" --- p.139 / Chapter 5.1.2 --- miRNA Profiling of DU145 --- p.139 / Chapter 5.1.3 --- "Confirming Induction of miR-765 by ICI 182,780 and ERβ dependency with qRT-PCR" --- p.140 / Chapter 5.1.4 --- "Up-regulation of miR-765 Host Gene, ARHGEF11, by ICI" --- p.141 / Chapter 5.2 --- Regulatory Elements of miR-765 Expression --- p.143 / Chapter 5.2.1 --- Own Upstream promoter of miR- 765 --- p.144 / Chapter 5.2.2 --- Promoter of Host Gene ARHGEF11 --- p.146 / Chapter 5.2.3 --- Interaction between ARHGEF11 Promoter Critical Region and Transcription Factors --- p.147 / Chapter 5.2.4 --- Involvement of independent Promoter and Host Gene Promoter in miR-765 Regulation --- p.757 / Chapter 5.3 --- Biological Significances of miR-765 on DU145 --- p.153 / Chapter 5.4 --- Significance of Findings and Future Studies --- p.158 / Chapter 5.4.1 --- Clinical Significance --- p.158 / Chapter 5.4.2 --- Future Studies --- p.161 / Chapter Chapter 6: --- Conclusion --- p.163 / Chapter Chapter 7: --- References --- p.166