Ng, Hin Yi Winnie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 137-140). / Abstracts in English and Chinese. / Acknowledgements --- p.ii / List of Tables --- p.iii / List of Figures --- p.iv / Abstract in English --- p.vi / Abstract in Chinese --- p.ix / Table of Contents --- p.xi / Chapter CHAPTER 1: --- INTRODUCTION --- p.1 / Chapter 1.1 --- Medulloblastoma (MB) --- p.1 / Chapter 1.1.1 --- Definition of Medulloblastoma --- p.1 / Chapter 1.1.2 --- Pathological Classification --- p.2 / Chapter 1.1.3 --- Current Treatment --- p.3 / Chapter 1.1.4 --- Molecular Pathology --- p.4 / Chapter 1.1.5 --- Molecular Classification of MB --- p.7 / Chapter 1.2 --- MicroRNAs (miRNAs) --- p.9 / Chapter 1.2.1 --- Biogenesis --- p.9 / Chapter 1.2.2 --- Functions --- p.10 / Chapter 1.2.3 --- MicroRNAs & Cancers --- p.10 / Chapter 1.2.4 --- Aberrant Expressions of MicroRNAs in Medulloblastoma --- p.12 / Chapter 1.2.5 --- MiR-106b-25 Cluster in MB --- p.13 / Chapter 1.2.6 --- miR-106b-25 Cluster in Regulating Target Genes --- p.15 / Chapter 1.2.7 --- Application of Regulatory miRNAs --- p.16 / Chapter 1.3 --- Target Gene Identification --- p.18 / Chapter 1.3.1 --- Recent Molecular Advances in Target Gene Identification --- p.18 / Chapter 1.3.2 --- Importance of Target Gene Identification --- p.19 / Chapter CHAPTER 2: --- AIMS OF STUDY --- p.21 / Chapter CHAPTER 3: --- COMPUTATIONAL TARGET PREDICTION --- p.23 / Chapter 3.1 --- Introduction- Computational Approach --- p.23 / Chapter 3.2 --- Methods --- p.27 / Chapter 3.2.1 --- Prediction Algorithms --- p.27 / Chapter 3.2.1.1 --- EIMMo2 --- p.27 / Chapter 3.2.1.2 --- miRDB --- p.27 / Chapter 3.2.1.3 --- miR-Tar-miRanda --- p.28 / Chapter 3.2.1.4 --- miR-Tar-RNAhybrid --- p.28 / Chapter 3.2.1.5 --- Diana-microT --- p.29 / Chapter 3.2.1.6 --- Pic-Tar --- p.29 / Chapter 3.2.1.7 --- TargetScan 4.2 --- p.29 / Chapter 3.2.2 --- Cell Culture --- p.30 / Chapter 3.2.2.1 --- Cell Lines --- p.30 / Chapter 3.2.2.2 --- Cell Counts --- p.31 / Chapter 3.2.3 --- Transfections --- p.31 / Chapter 3.2.3.1 --- Transfection of MicroRNA Inhibitors --- p.31 / Chapter 3.2.3.1.1 --- Transfection Efficiency of Lipofectamine2000 --- p.32 / Chapter 3.2.3.1.2 --- Transfection of MicroRNA Inhibitors for Real-time PCR --- p.32 / Chapter 3.2.3.1.3 --- Transfection of MicroRNA Inhibitors for Western Blotting --- p.33 / Chapter 3.2.3.2 --- Co-transfection of Plasmid and MicroRNA Inhibitors --- p.33 / Chapter 3.2.3.2.1 --- Blocking Efficiency of MicroRNA Inhibitors --- p.33 / Chapter 3.2.3.2.2 --- Co-transfection of Target Gene Expression Vector and MicroRNA Inhibitors --- p.34 / Chapter 3.2.4 --- Real-time PCR Amplification --- p.35 / Chapter 3.2.4.1 --- Total RNA Extraction from Cell Lines --- p.35 / Chapter 3.2.4.2 --- Stemloop miRNA Taqman qRT-PCR Analysis --- p.36 / Chapter 3.2.4.3 --- Reverse Transcription --- p.37 / Chapter 3.2.4.4 --- Real-time PCR Target Gene Expression --- p.38 / Chapter 3.2.5 --- Cloning of Potential Target Genes into pMIR Luciferase Expression Vector --- p.39 / Chapter 3.2.5.1 --- High-Fidelity PCR Amplification of yUTRs --- p.41 / Chapter 3.2.5.2 --- PCR Purification of Amplified PCR Product --- p.42 / Chapter 3.2.5.3 --- Restriction Enzyme Digestions --- p.42 / Chapter 3.2.5.4 --- Ligation of 3'UTR to Expression Vector --- p.43 / Chapter 3.2.5.5 --- Transformation --- p.43 / Chapter 3.2.5.6 --- Preparation of the Cloned Plasmid --- p.43 / Chapter 3.2.5.7 --- Sequencing of the Cloned Plasmid --- p.44 / Chapter 3.2.6 --- Site-directed Mutagenesis --- p.45 / Chapter 3.2.7 --- Dual-Luciferase Assay --- p.47 / Chapter 3.2.8 --- Western Blot Analysis --- p.47 / Chapter 3.3 --- Results --- p.49 / Chapter 3.3.1 --- Expression Levels of miR-106b-25 Cluster in MB Cell Lines --- p.49 / Chapter 3.3.2 --- Evaluation of Transfection Efficiency Using Lipofetamine2000 --- p.51 / Chapter 3.3.3 --- Blocking Efficiency of MicroRNA Inhibitors --- p.52 / Chapter 3.3.4 --- Target Prediction List --- p.53 / Chapter 3.3.5 --- Recognition Sites of Potential Targets --- p.55 / Chapter 3.3.6 --- Expression Levels of ZNFX1 in MB Cell Lines --- p.56 / Chapter 3.3.7 --- Transcriptional Regulation of ZNFXl and DNAJB12 --- p.57 / Chapter 3.3.8 --- Verification of Potential Target Genes --- p.59 / Chapter 3.3.9 --- Identification of Critical Target Sites --- p.61 / Chapter 3.3.10 --- Effects of Anti-microRNA Inhibitors on ZNFX1 Protein Levels --- p.66 / Chapter 3.4 --- Discussion --- p.67 / Chapter CHAPTER 4: --- EXPERIMENTAL APPROACH IN INDENTIFYING POTENTIAL TARGETS --- p.77 / Chapter 4.1 --- Introduction- Experimental Approach --- p.74 / Chapter 4.2 --- Methods --- p.79 / Chapter 4.2.1 --- Isolation of cDNA Clone Library --- p.79 / Chapter 4.2.1.1 --- Preparation of Cytoplasmic Extracts --- p.79 / Chapter 4.2.1.2 --- Reverse Transcription Using Endogenous miRNA as Primers --- p.81 / Chapter 4.2.1.3 --- Collection of Polynucleotides --- p.82 / Chapter 4.2.1.4 --- Synthesis of Second-strand cDNAs --- p.82 / Chapter 4.2.1.5 --- PCR Purification of Double-stranded cDNAs --- p.83 / Chapter 4.2.1.6 --- Restriction Endonuclease Digestion --- p.84 / Chapter 4.2.1.7 --- Ligation to Adaptor --- p.85 / Chapter 4.2.1.8 --- PCR Amplification with Biotin-labelled miRNA PCR Primers --- p.86 / Chapter 4.2.1.9 --- Capture of Biotin-labelled PCR Fragments --- p.88 / Chapter 4.2.1.10 --- Introducing NotI Recognition Sequences --- p.88 / Chapter 4.2.1.11 --- Cloning into the pCR2.1 Vector --- p.89 / Chapter 4.2.1.12 --- Ligation of the cDNA Fragments and the pCR2.1 Vector --- p.90 / Chapter 4.2.1.13 --- Transformation --- p.90 / Chapter 4.2.1.14 --- Preparation of Purified Plasmids --- p.91 / Chapter 4.2.1.15 --- Sequencing Analysis of the cDNA Clone Library --- p.91 / Chapter 4.2.2 --- Real-time PCR Target Gene Expression in Cell Lines --- p.92 / Chapter 4.2.3 --- Real-time PCR Target Gene Expression Upon Inhibition of miR-106b --- p.92 / Chapter 4.2.4 --- Cloning of Potential Target Genes into pMIR Luciferase Expression Vector --- p.93 / Chapter 4.2.5 --- Site-directed Mutagenesis --- p.94 / Chapter 4.2.6 --- Luciferase Reporter Assay --- p.94 / Chapter 4.3 --- Results --- p.95 / Chapter 4.3.1 --- Sequencing Analysis of the cDNA Clone Library --- p.95 / Chapter 4.3.2 --- Expression Levels of Candidate Genes in MB Cell Lines --- p.100 / Chapter 4.3.3 --- Effects of Anti-miR-106b Inhibitors on 3'UTR of Target Genes --- p.101 / Chapter 4.3.4 --- Verification of Candidate Genes --- p.103 / Chapter 4.3.5 --- Verification of Target Sites with Site-directed Mutagenesis --- p.104 / Chapter 4.4 --- Discussion --- p.107 / Chapter CHAPTER 5: --- FUNCTIONAL ASSAYS --- p.111 / Chapter 5.1 --- Introduction- Functional Investigation of miR-106b-25 Cluster --- p.111 / Chapter 5.2 --- Methods --- p.113 / Chapter 5.2.1 --- Cell Culture --- p.113 / Chapter 5.2.2 --- Over-expression of miR-106b Mimic --- p.113 / Chapter 5.2.3 --- MTT Assay --- p.114 / Chapter 5.2.4 --- IC50 of Cisplatin --- p.115 / Chapter 5.2.5 --- MTT Assay with Cisplatin Treatment --- p.115 / Chapter 5.2.6 --- Cell Cycle --- p.116 / Chapter 5.2.7 --- BrdU Cell Proliferation Assay --- p.117 / Chapter 5.2.8 --- Wound Healing Assay --- p.117 / Chapter 5.3 --- Results --- p.119 / Chapter 5.3.1 --- Effects of Inhibition of miR-106b-25 Cluster on Cell Growth. --- p.119 / Chapter 5.3.2 --- Cell Cycle Distribution Analysis --- p.121 / Chapter 5.3.3 --- Sensitivity to Cisplatin --- p.123 / Chapter 5.3.4 --- Cell Proliferation Assay --- p.124 / Chapter 5.3.5 --- Cell Motility --- p.126 / Chapter 5.3.6 --- Efficiency of Over-expression Using miR-106b Mimic --- p.129 / Chapter 5.3.7 --- Effects of miR-106b on Cell Growth --- p.130 / Chapter 5.4 --- Discussion --- p.131 / Chapter CHAPTER 6: --- CONCLUSION --- p.135 / REFERENCE --- p.137
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_327461 |
| Date | January 2011 |
| Contributors | Ng, Hin Yi Winnie., Chinese University of Hong Kong Graduate School. Division of Anatomical and Cellular Pathology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xvi, 140 leaves : ill. ; 30 cm. |
| Rights | Use 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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