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Molecular and functional characterization of microRNA-137 in oligodendroglial tumors.

Yang, Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 222-244). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Awards and Presentations --- p.ii / Abstract in English --- p.iii / Abstract in Chinese --- p.vii / Table of Contents --- p.x / List of Tables --- p.xv / List of Figures --- p.xvii / List of Abbreviations --- p.xx / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Gliomas --- p.1 / Chapter 1.1.1 --- Oligodendroglial tumors (OTs) --- p.3 / Chapter 1.1.2 --- Glioblastoma multiforme (GBM) --- p.3 / Chapter 1.1.3 --- Molecular pathology of gliomas --- p.4 / Chapter 1.1.3.1 --- Genetic alterations in OTs --- p.4 / Chapter 1.1.3.2 --- Prognostic and predictive factors in OTs --- p.7 / Chapter 1.1.3.3 --- Genetic alterations in GBM --- p.8 / Chapter 1.1.3.4 --- Prognostic and predictive factors in GBM --- p.10 / Chapter 1.2 --- microRNA(miRNA) --- p.13 / Chapter 1.2.1 --- miRNA biogenesis and function --- p.13 / Chapter 1.2.2 --- miRNA involvement in cancer --- p.17 / Chapter 1.2.2.1 --- Dysregulation of miRNAs in human malignancies --- p.17 / Chapter 1.2.2.2 --- Function and potential application of miRNAs --- p.17 / Chapter 1.2.3 --- Role of miRNAs in glioma --- p.19 / Chapter 1.2.3.1 --- miRNAs in OTs --- p.19 / Chapter 1.2.3.2 --- miRNAs in GBM --- p.20 / Chapter 1.3 --- miR-137 --- p.30 / Chapter 1.3.1 --- Biology of miR-137 --- p.30 / Chapter 1.3.2 --- Role of miR-137 in carcinogenesis --- p.33 / Chapter 1.3.2.1 --- Deregulation of miR-137 in cancer --- p.33 / Chapter 1.3.2.2 --- Regulation of miR-137 expression in cancer --- p.33 / Chapter 1.3.2.3 --- Biological functions of miR-137 in cancer --- p.37 / Chapter 1.3.3 --- Role of miR-137 in differentiation and neurogenesis --- p.39 / Chapter CHAPTER 2 --- AIMS OF STUDY --- p.43 / Chapter CHARPTER 3 --- MATERIALS AND METHODS --- p.45 / Chapter 3.1 --- Tumor samples --- p.45 / Chapter 3.2 --- Cell lines and culture conditions --- p.48 / Chapter 3.3 --- Fluorescence in situ hybridization (FISH) --- p.49 / Chapter 3.4 --- Cell transfection --- p.52 / Chapter 3.4.1 --- Transfection of oligonucleotides --- p.52 / Chapter 3.4.1.1 --- Oligonucleotide preparation --- p.52 / Chapter 3.4.1.2 --- Optimization of transfection condition --- p.52 / Chapter 3.4.2 --- Cotransfection of plasmids and miRNA mimic --- p.53 / Chapter 3.4.2.1 --- Optimization of transfection condition --- p.53 / Chapter 3.4.2.2 --- Procedure of transfection --- p.54 / Chapter 3.5 --- Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) --- p.55 / Chapter 3.5.1 --- RNA extraction from frozen tissues and cell lines --- p.55 / Chapter 3.5.2 --- qRT-PCR for miR-137 --- p.56 / Chapter 3.5.3 --- qRT-PCR for CSE1L and ERBB4 transcripts --- p.57 / Chapter 3.6 --- 5-aza-2'-deoxycytidine (5-aza-dC) and Trichostatin A (TSA) treatment --- p.61 / Chapter 3.7 --- Western blotting --- p.62 / Chapter 3.7.1 --- Preparation of cell lysate --- p.62 / Chapter 3.7.2 --- Measurement of protein concentration --- p.62 / Chapter 3.7.3 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.63 / Chapter 3.7.4 --- Electroblotting of proteins --- p.67 / Chapter 3.7.5 --- Immunoblotting --- p.67 / Chapter 3.8 --- Dual-luciferase reporter assay --- p.70 / Chapter 3.8.1 --- Construction of reporter plasmids --- p.70 / Chapter 3.8.1.1 --- Experimental outline --- p.70 / Chapter 3.8.1.2 --- PCR Amplification of MREs --- p.70 / Chapter 3.8.1.3 --- TA cloning --- p.71 / Chapter 3.8.1.4 --- Transformation --- p.72 / Chapter 3.8.1.5 --- Blue/white screening and validation of recombinants --- p.72 / Chapter 3.8.1.6 --- Subcloning of 3'UTR fragments into pMIR-reproter vector --- p.73 / Chapter 3.8.2 --- Site-directed mutagenesis --- p.74 / Chapter 3.8.3 --- Plasmid and miRNA mimic cotransfection --- p.76 / Chapter 3.8.4 --- Determination of luciferase activity --- p.76 / Chapter 3.9 --- Functional assays : --- p.79 / Chapter 3.9.1 --- Cell growth and proliferation assay --- p.79 / Chapter 3.9.1.1 --- "3-(4,5-Dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay" --- p.79 / Chapter 3.9.1.2 --- Cell counting --- p.80 / Chapter 3.9.1.3 --- 5-Bromo-2'-deoxyuridine (BrdU) incorporation assay --- p.80 / Chapter 3.9.2 --- Apoptosis assay --- p.82 / Chapter 3.9.3 --- Anchorage-independent growth assay --- p.82 / Chapter 3.9.4 --- Wound healing assay --- p.83 / Chapter 3.9.5 --- Matrigel invasion assay --- p.84 / Chapter 3.9.6 --- Cell differentiation assay --- p.85 / Chapter 3.10 --- Immunohistochemical analysis --- p.86 / Chapter 3.10.1 --- H&E staining --- p.86 / Chapter 3.10.2 --- Detection of Ki-67 expression --- p.87 / Chapter 3.10.3 --- Detection of CSE1L expression --- p.87 / Chapter 3.10.4 --- Scoring methods --- p.88 / Chapter 3.11 --- Bioinformatic analysis --- p.90 / Chapter 3.12 --- Statistical analysis --- p.92 / Chapter CHAPTER 4 --- RESULTS --- p.93 / Chapter 4.1 --- Expression of miR-137 in glioma cells and clinical significance --- p.93 / Chapter 4.1.1 --- Description of 36 OT samples --- p.93 / Chapter 4.1.2 --- miR-137 level in oligodendroglial tumors and glioma cells --- p.102 / Chapter 4.1.3 --- "Association of miR-137 expression with clinicopathological features, lp/19q status and Ki-67 expression" --- p.104 / Chapter 4.2 --- miR-137 levels in glioma cells after demethylation treatment --- p.113 / Chapter 4.3 --- Biological effects of miR-137 overexpression in glioma cells --- p.118 / Chapter 4.3.1 --- Cell growth --- p.118 / Chapter 4.3.1.1 --- Cell viability --- p.118 / Chapter 4.3.1.2 --- Cell number --- p.123 / Chapter 4.3.1.3 --- Cell cycle analysis : --- p.127 / Chapter 4.3.2 --- Anchorage-independent cell growth --- p.130 / Chapter 4.3.3 --- Cell apoptosis --- p.134 / Chapter 4.3.4 --- Cell motility --- p.136 / Chapter 4.3.5 --- Cell differentiation : --- p.142 / Chapter 4.4 --- Identification of miR-137 targets --- p.144 / Chapter 4.4.1 --- In silico prediction of potential miR-137 targets --- p.144 / Chapter 4.4.2 --- Experimental validation of miR-137 targets by dual-luciferase reporter assay --- p.147 / Chapter 4.4.3 --- "Expression of miR-137 candidate targets, CSE1L and ERBB4 in glioma cells" --- p.152 / Chapter 4.4.4 --- Effects of miR-137 on CSE1L transcript and protein levels --- p.154 / Chapter 4.5 --- Expression of CSE1L in OTs --- p.156 / Chapter 4.5.1 --- CSE1L expression in OTs by qRT-PCR and IHC --- p.156 / Chapter 4.5.2 --- Correlation of CSE1L expression with clinicopathological features --- p.165 / Chapter 4.6 --- Effects of CSE1L knockdown in glioma cells --- p.168 / Chapter 4.6.1 --- Cell growth --- p.170 / Chapter 4.6.1.1 --- Cell viability --- p.170 / Chapter 4.6.1.2 --- Cell number --- p.173 / Chapter 4.6.1.3 --- Cell cycle analysis --- p.176 / Chapter 4.6.2 --- Anchorage-independent cell growth --- p.179 / Chapter 4.6.3 --- Cell apoptosis --- p.182 / Chapter 4.6.4 --- Cell motility --- p.184 / Chapter CHAPTER 5 --- DISCUSSION --- p.190 / Chapter 5.1 --- Expression of miR-137 transcript level in OTs and glioma cell lines --- p.190 / Chapter 5.2 --- Association of miR-137 expression with OT clinical and molecular parameters --- p.192 / Chapter 5.3 --- Prognostic significance of clinical features and miR-137 expression in OTs --- p.194 / Chapter 5.4 --- Inactivation mechanisms of miR-137 in glioma --- p.196 / Chapter 5.5 --- Biological effects of miR-137 overexpression in glioma cells --- p.198 / Chapter 5.6 --- CSE1L is a novel miR-137 target in glioma --- p.200 / Chapter 5.7 --- Expression of CSE1L in glioma --- p.203 / Chapter 5.8 --- Intracellular distribution of CSElL in OTs --- p.206 / Chapter 5.9 --- Correlation of CSE1L expression with clinicopathological and molecular features in OTs --- p.208 / Chapter 5.10 --- CSE1L mediates effects of miR-137 in glioma cells --- p.210 / Chapter 5.11 --- Biological roles of CSE1L in glioma cells 226}0Ø. --- p.212 / Chapter 5.11.1 --- CSE1L in glioma cell proliferation --- p.212 / Chapter 5.11.2 --- CSE1L in glioma cell apoptosis --- p.213 / Chapter 5.11.3 --- CSE1L in glioma cell invasion --- p.215 / Chapter CHAPTER 6 --- CONCLUSIONS --- p.216 / Chapter CHAPTER 7 --- FUTURE STUDIES --- p.219 / Chapter 7.1 --- Expression Molecular mechanisms for miR-137 inactivation in glioma --- p.219 / Chapter 7.2 --- Identification of more miR-137 targets in glioma --- p.219 / Chapter 7.3 --- Role of miR-137 and CSE1L in drug-induced apoptosis in glioma --- p.220 / Chapter 7.4 --- Deciphering dysregulated and clinical relevant miRNAs in glioma --- p.220 / Chapter 7.5 --- Effects of miR-137 in vivo and the therapeutic potential in glioma treatment --- p.221 / REFERENCES --- p.222

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_327460
Date January 2011
ContributorsYang, Ling., Chinese University of Hong Kong Graduate School. Division of Anatomical and Cellular Pathology.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xxiv, 244 leaves : ill. (some col.) ; 30 cm.
RightsUse of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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