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Epigenetic identification of novel 12p and 16q tumor suppressor genes for multiple carcinomas.January 2007 (has links)
Lee, Kwan Yeung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 103-113). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.v / Table of Content --- p.vi / List of Figures --- p.xi / List of Tables --- p.xiii / List of Abbreviations --- p.xiv / List of papers published during the study --- p.xvi / Chapter Chapter 1 --- Introduction and Aim of Study --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Project objective and potential significances --- p.5 / Chapter Chapter 2 --- Literatures Review --- p.6 / Chapter 2.1 --- Cancer genetics and Tumor suppressor genes --- p.6 / Chapter 2.2 --- Epigenetic --- p.7 / Chapter 2.2.1 --- DNA methylation and promoter CpG island --- p.8 / Chapter 2.2.2 --- Establishment and maintenance of DNA methylation --- p.9 / Chapter 2.2.3 --- Transcriptional silencing by DNA hypermethylation --- p.9 / Chapter 2.3 --- Cancer epigenetic --- p.11 / Chapter 2.3.1 --- Hypomethylation of the cancer genome --- p.12 / Chapter 2.3.2 --- Hypermethylation in cancers --- p.12 / Chapter 2.3.3 --- Clinical relevance of cancer epigenetic --- p.13 / Chapter 2.4 --- Nasopharyngeal carcinoma --- p.14 / Chapter 2.4.1 --- NPC genetic and epigenetic --- p.15 / Chapter 2.5 --- 12p as a putative tumor suppressor locus --- p.16 / Chapter 2.5.1 --- Hematological malignancies associated with 12p loss --- p.17 / Chapter 2.5.2 --- Prostate cancer associated with 12p loss --- p.20 / Chapter 2.5.3 --- Lung cancer associated with 12p loss --- p.22 / Chapter 2.5.4 --- 12p deletion in other cancers --- p.23 / Chapter 2.6 --- 16q as a tumor suppressor locus --- p.24 / Chapter 2.6.1 --- Breast cancer and 16q --- p.25 / Chapter 2.6.2 --- Loss of 16q and prostate cancer --- p.26 / Chapter 2.6.3 --- Loss of 16q and hepatocellular carcinoma --- p.28 / Chapter 2.6.4 --- 16q deletion associated with other cancers --- p.29 / Chapter Chapter 3 --- Materials and Methods --- p.30 / Chapter 3.1 --- Cell lines and tissue samples --- p.30 / Chapter 3.1.1 --- Cell lines --- p.30 / Chapter 3.1.2 --- Maintenance of cell lines --- p.31 / Chapter 3.1.3 --- Drugs treatment of cell lines --- p.31 / Chapter 3.1.4 --- Normal tissues --- p.32 / Chapter 3.1.5 --- Total RNA extraction --- p.32 / Chapter 3.1.6 --- Genomic DNA extraction --- p.32 / Chapter 3.2 --- General techniques --- p.33 / Chapter 3.2.2 --- TA cloning and blunt end cloning of PCR product --- p.33 / Chapter 3.2.3 --- Transformation of cloning products to E. coli competent cells --- p.34 / Chapter 3.2.4 --- Preparation of plasmid DNA --- p.34 / Chapter 3.2.4.1 --- Mini-prep plasmid DNA extraction --- p.34 / Chapter 3.2.4.2 --- Midi-prep of plasmid DNA --- p.35 / Chapter 3.2.5 --- Measurement of DNA or RNA concentrations --- p.36 / Chapter 3.2.6 --- DNA sequencing of plasmid DNA and PCR products --- p.36 / Chapter 3.3 --- Preparation of reagents and medium --- p.37 / Chapter 3.4 --- Semi-quantitative Reverse-Transcription (RT) PCR expression analysis --- p.38 / Chapter 3.4.1 --- Reverse transcription reaction --- p.38 / Chapter 3.4.2 --- Semi-quantitative RT-PCR --- p.39 / Chapter 3.4.2.1 --- Primers design --- p.39 / Chapter 3.4.2.2 --- PCR reaction --- p.39 / Chapter 3.5 --- Methylation analysis of candidate genes --- p.40 / Chapter 3.5.1 --- Bisulfite treatment of genomic DNA --- p.41 / Chapter 3.5.2 --- Methylation-specific PCR (MSP) --- p.42 / Chapter 3.5.2.1 --- Bioinformatics prediction of CpG island --- p.42 / Chapter 3.5.2.2 --- Primers design --- p.42 / Chapter 3.5.2.3 --- PCR reaction --- p.42 / Chapter 3.5.3 --- Bisulfite Genomic Sequencing (BGS) --- p.43 / Chapter 3.5.3.1 --- Primers design --- p.43 / Chapter 3.5.3.2 --- PCR reaction --- p.44 / Chapter 3.6 --- Construction of expression vectors of candidate genes --- p.44 / Chapter 3.6.1 --- Construction of IRF8 expression vector --- p.44 / Chapter 3.6.2 --- Construction of PTPRO expression vector --- p.44 / Chapter 3.6.2.1 --- Experimental design --- p.44 / Chapter 3.6.2.2 --- PCR and cloning of PCR products --- p.46 / Chapter 3.6.2.3 --- Restriction digestion of cloning vectors and expression vector --- p.48 / Chapter 3.6.2.4 --- Ligation of cloning fragments --- p.48 / Chapter 3.7 --- Colony formation assay on monolayer culture --- p.48 / Chapter 3.8 --- Statistical analysis --- p.49 / Chapter Chapter 4 --- Identification of candidate TSGs in deleted regions --- p.50 / Chapter 4.1 --- Research plan --- p.50 / Chapter 4.2 --- Results --- p.50 / Chapter 4.2.1 --- Mapping of the deleted B AC clones on their chromosomal locations --- p.50 / Chapter 4.2.2 --- Identification of down-regulated genes in NPC by semi-quantitative RT-PCR analysis --- p.51 / Chapter 4.3 --- Discussion --- p.55 / Chapter Chapter 5 --- Tumor suppressor function studies of candidate TSGs --- p.60 / Chapter 5.1 --- Research plan --- p.60 / Chapter 5.2. --- IRF8 is the 16q candidate TSG --- p.60 / Chapter 5.2.1 --- Frequent silencing of IRF8 mRNA expression in multiple carcinomas --- p.60 / Chapter 5.2.2 --- Methylation status of IRF8 promoter region correlated with its transcriptional silencing --- p.62 / Chapter 5.2.3 --- Restoration of IRF8 expression by pharmacological and genetic demethylation --- p.65 / Chapter 5.2.4 --- IRF8 inhibited the anchorage dependent growth of tumor cells on monolayer culture --- p.67 / Chapter 5.2.5 --- Discussion --- p.68 / Chapter 5.3 --- PTPRO is the down-regulated target at 12pl3.2-12.3 tumor suppressor locus --- p.73 / Chapter 5.3.1 --- Frequent silencing of PTPRO in multiple carcinoma cell lines --- p.73 / Chapter 5.3.2 --- Frequent methylation of PTPRO promoter CpG island in multiple carcinoma cell lines correlated with its reduced expression --- p.74 / Chapter 5.3.3 --- Re-expression of PTPRO by pharmacological and genetic demethylation --- p.77 / Chapter 5.3.4 --- PTPRO inhibited the growth of tumor cells in vitro --- p.79 / Chapter 5.3.5 --- Discussion --- p.81 / Chapter 5.4 --- RERG is another candidate TSG in 12pl3.2 - 12.3 region --- p.87 / Chapter 5.4.1 --- Down-regulation of RERG mRNA expression in carcinoma cell line --- p.87 / Chapter 5.4.2 --- Hypermethylation of RERG promoter is a frequent event in multiple carcinomas --- p.88 / Chapter 5.4.3 --- Re-expression of RERG mRNA following pharmacological and genetic demethylation --- p.90 / Chapter 5.4.4 --- Discussion --- p.92 / Chapter Chapter 6 --- General discussion --- p.96 / Chapter Chapter 7 --- Summary --- p.101 / Reference --- p.103
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Identification of novel candidate tumor suppressor genes at 5q and 14q for multiple carcinomas by integrative genomics and epigenetics.January 2007 (has links)
Ng, Ka Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 103-113). / Abstracts in English and Chinese. / Acknowledgements --- p.i / List of abbreviations --- p.ii / List of Tables --- p.iv / List of Figures --- p.v / List of Publications --- p.viii / Abstract in English --- p.ix / Abstract in Chinese --- p.xi / Table of Contents --- p.xiii / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Tumor suppressor genes (TSGs) and the modes of TSG inactivation during carcinogenesis --- p.1 / Chapter 1.2 --- Epigenetic modifications --- p.3 / Chapter 1.2.1 --- DNA methylation --- p.4 / Chapter 1.2.1a --- Establishment of DNA methylation patterns and DNA methyltransferases --- p.5 / Chapter 1.2.1b --- DNA hypermethylation and carcinogenesis --- p.6 / Chapter 1.2.1c --- Mechanism for gene silencing by CpG methylation --- p.6 / Chapter 1.2.1d --- DNA hypomethylation and carcinogenesis --- p.10 / Chapter 1.2.1e --- Loss of imprinting and carcinogenesis --- p.11 / Chapter 1.2.1f --- Potential factors leading to aberrant methylation patterns in cancers --- p.12 / Chapter 1.2.2 --- Deregulation of histone modifications and carcinogenesis --- p.14 / Chapter 1.2.3 --- Interplay between chromatin modifications and DNA methylation --- p.15 / Chapter 1.3 --- Identification of tumor suppressor genes (TSGs) --- p.17 / Chapter 1.4 --- Nasopharyngeal carcinoma as a cancer model of the current project --- p.18 / Chapter 1.5 --- Genetic and epigenetic changes in NPC --- p.19 / Chapter 1.6 --- Involvement of 5qll-ql2 and 14q32 in carcinogenesis --- p.22 / Chapter 1.6.1 --- Chromosome 5ql l-ql2 and carcinogenesis --- p.22 / Chapter 1.6.2 --- Chromosome 14q32 and carcinogenesis --- p.24 / Chapter 1.7 --- Clinical implications of epigenetics in cancers --- p.27 / Chapter Chapter 2 --- Aims of study and Research plan --- p.31 / Chapter Chapter 3 --- Materials and Methods --- p.34 / Chapter 3.1 --- Cell lines and Normal Tissues --- p.35 / Chapter 3.2 --- Routine cell line maintenance --- p.35 / Chapter 3.3 --- Drug treatments --- p.35 / Chapter 3.4 --- Total RNA extraction --- p.35 / Chapter 3.5 --- Genomic DNA extraction --- p.36 / Chapter 3.6 --- General techniques --- p.37 / Chapter 3.6.1 --- Gel electrophoresis --- p.37 / Chapter 3.6.2 --- DNA and RNA quantification --- p.37 / Chapter 3.6.3 --- LB medium and LB plate preparation --- p.38 / Chapter 3.6.4 --- Plasmid extraction --- p.38 / Chapter 3.6.4a --- Mini-scale preparation of plasmid DNA --- p.38 / Chapter 3.6.4b --- Large-scale preparation of endotoxin-free plasmid DNA --- p.39 / Chapter 3.6.5 --- DNA sequencing --- p.39 / Chapter 3.7 --- Reverse transcription-PCR (RT-PCR) --- p.40 / Chapter 3.7.1 --- Reverse transcription (RT) --- p.40 / Chapter 3.7.2 --- Semi-quantitative RT-PCR --- p.41 / Chapter 3.8 --- Methylation analysis --- p.42 / Chapter 3.8.1 --- Sodium bisulfite modification of DNA --- p.42 / Chapter 3.8.2 --- CpG island analysis --- p.42 / Chapter 3.8.3 --- Methylation-specific PCR (MSP) --- p.43 / Chapter 3.8.4 --- Bisulfite genomic sequencing (BGS) --- p.44 / Chapter 3.9 --- Construction of expression plasmids --- p.45 / Chapter 3.9.1 --- Construction of the MGC80-expressing vector --- p.45 / Chapter 3.9.2 --- Construction of the TUSC14-expressing vector --- p.46 / Chapter 3.10 --- Functional analyses --- p.47 / Chapter 3.10.1 --- Monolayer colony formation assay --- p.47 / Chapter 3.10.2 --- Soft agar assay --- p.48 / Chapter 3.11 --- Statistical analysis --- p.49 / Chapter Chapter 4 --- Results --- p.50 / Chapter 4.1 --- Identification of 5qll-ql2 and 14q32.2-q32.32 as frequently deleted regions in NPC by aCGH --- p.50 / Chapter 4.2 --- Identification of novel candidate TSGs at chromosome 5qll-ql2 through integrative genomics and epigenetics --- p.51 / Chapter 4.2.1 --- Expression profiling of the candidate genes at 5ql l-ql2 in NPC cell lines --- p.51 / Chapter 4.2.2 --- MGC80 as a target of study at 5ql2 --- p.54 / Chapter 4.2.2a --- Ubiquitous expression in normal human tissues and frequent down-regulation of MGC80 in multiple tumor cell lines --- p.54 / Chapter 4.2.2b --- Methylation analysis of MGC80 --- p.56 / Chapter 4.2.2c --- Restoration of MGC80 expression after pharmacologic and genetic demethylation --- p.59 / Chapter 4.2.2d --- Functional study of MGC80 in multiple carcinomas --- p.61 / Chapter 4.2.2e --- Discussion --- p.63 / Chapter 4.2.3 --- TUSC14 as a target of study at 5ql2 --- p.67 / Chapter 4.2.3a --- TUSC14 was broadly expressed in normal human tissues and frequently down-regulated in multiple tumor cell lines --- p.67 / Chapter 4.2.3b --- Methylation analysis of TUSCI4 --- p.69 / Chapter 4.2.3c --- Pharmacologic and genetic demethylation reactivated TUSC14 expression --- p.72 / Chapter 4.2.3d --- Functional study ofTUSC14 in multiple carcinomas --- p.74 / Chapter 4.2.3e --- Discussion --- p.76 / Chapter 4.3 --- Identification of candidate TSGs at chromosome 14q32 through integrative genomics and epigenetics --- p.80 / Chapter 4.3.1 --- Expression profiling of the candidate genes at 14q32 in NPC cell lines --- p.80 / Chapter 4.3.2 --- DLK1 as a target of study at 14q32 --- p.82 / Chapter 4.3.2a --- Expression analysis of DLK1 in normal tissues and NPC cell lines --- p.82 / Chapter 4.3.2b --- Methylation analysis ofDLKl in NPC --- p.83 / Chapter 4.3.2c --- Restoration of DLK1 expression after pharmacologic demethylation --- p.84 / Chapter 4.3.2d --- Functional study ofDLKl in NPC --- p.85 / Chapter 4.3.2e --- Discussion --- p.87 / Chapter Chapter 5 --- General discussion --- p.92 / Chapter Chapter 6 --- Summary --- p.99 / Chapter Chapter 7 --- Future study --- p.101 / Reference list --- p.103
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