Diao, Shu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 72-73). / Abstracts in English and Chinese. / 摘要 --- p.i / Abstract --- p.ii / Acknowledgements --- p.iv / Contents --- p.viii / Chapter Chapter 1 --- Introduction and Background / Chapter 1.1 --- General introduction to miRNA --- p.1 / Chapter 1.1.1 --- The discovery and biogenesis of miRNA --- p.1 / Chapter 1.1.2 --- The function of miRNA --- p.3 / Chapter 1.2 --- The liver-specific miRNA : miR-122a --- p.5 / Chapter 1.2.1 --- Discovery and biogenesis of miR-122a --- p.5 / Chapter 1.2.2 --- "miR-122a, a liver specific miRNA" --- p.6 / Chapter 1.2.3 --- miR-122a and Hepatocellular carcinoma (HCC) --- p.6 / Chapter 1.2.4 --- Therapeutic opportunities and challenges of miR-122a --- p.7 / Chapter 1.3 --- Techniques and approaches to study miRNAs --- p.8 / Chapter 1.3.1 --- Discovery of novel miRNA --- p.8 / Chapter 1.3.2 --- Detection of miRNA --- p.9 / Chapter 1.3.3 --- Functional study of miRNAs --- p.10 / Chapter 1.3.4 --- Prediction and validation of miRNA targets --- p.10 / Chapter 1.4 --- References --- p.12 / Chapter Chapter 2 --- Materials and methods / Chapter 2.1 --- Cell culture --- p.18 / Chapter 2.2 --- Cell transfection --- p.18 / Chapter 2.3 --- RNA extraction --- p.18 / Chapter 2.4 --- Plasmid extaction --- p.19 / Chapter 2.5 --- Competent cell preparation --- p.19 / Chapter 2.6 --- Bacterial Transformation --- p.20 / Chapter 2.7 --- Purification of DNA fragments from agarose gel --- p.21 / Chapter 2.8 --- Genomic DNA extranction from MIHA cell --- p.21 / Chapter 2.9 --- Real-time RT-PCR analysis --- p.21 / Chapter 2.10 --- Lenti-vector Construction for miRNA expression --- p.22 / Chapter 2.11 --- Lentivirus production --- p.22 / Chapter 2.12 --- Lentiviral vector titering --- p.23 / Chapter 2.13 --- Bradford protein assay --- p.23 / Chapter 2.14 --- Western Blot --- p.24 / Chapter 2.14.1 --- Sample preparation --- p.24 / Chapter 2.14.2 --- Gel electrophoresis --- p.24 / Chapter 2.14.3 --- Blocking --- p.25 / Chapter 2.14.4 --- Incubation with Primary and Secondary Antibodies --- p.25 / Chapter 2.14.5 --- Substrate Incubation --- p.26 / Chapter 2.14.6 --- Exposeto x-ray film --- p.26 / Chapter 2.15 --- MTT Cell Proliferation Assay --- p.26 / Chapter 2.16 --- Apoptosis analysis :DAPI Staining --- p.26 / Chapter 2.17 --- 2-D Protein Gel Electrophoresis and MS --- p.26 / Chapter 2.17.1 --- Materials --- p.27 / Chapter 2.17.2 --- Protein extraction --- p.27 / Chapter 2.17.3 --- 2-D Electrophoresis --- p.27 / Chapter 2.17.4 --- Gel staining and image analysis --- p.28 / Chapter 2.17.5 --- In-gel protein digestion with trypsin --- p.28 / Chapter 2.17.6 --- MALDI-TOF MS and database search --- p.28 / Chapter 2.18 --- Statistical Analysis --- p.29 / Chapter Chapter 3 --- Expression of HCC-associated miRNA in HCC cell lines / Chapter 3.1 --- Introduction --- p.30 / Chapter 3.2 --- Experimental Section --- p.31 / Chapter 3.2.1 --- Cell culture --- p.31 / Chapter 3.2.2 --- RNA extraction --- p.31 / Chapter 3.2.3 --- miRNA-specific quantitative Real-time PCR --- p.31 / Chapter 3.3 --- Results and Discussion --- p.32 / Chapter 3.3.1 --- Expression of miR-let7a in HCC cells --- p.32 / Chapter 3.3.2 --- Expression of miR-221 in HCC cells --- p.33 / Chapter 3.3.3 --- "Expression of miR-122a,a liver-specific miRNA in HCC cells" --- p.34 / Chapter 3.4 --- Conclusions --- p.35 / Chapter 3.5 --- References --- p.36 / Chapter Chapter 4 --- Ectopic overexpression of miR-122a in HCC cells / Chapter 4.1 --- Introduction --- p.38 / Chapter 4.2 --- Experimental Section --- p.38 / Chapter 4.2.1 --- Overexpression of miR-122a with mimics --- p.38 / Chapter 4.2.2 --- Lentivirus-mediated miR-122a expression --- p.40 / Chapter 4.2.3 --- RNA extraction --- p.44 / Chapter 4.2.4 --- Expression level of miR-122a after transduction --- p.44 / Chapter 4.2.5 --- Western Blot --- p.44 / Chapter 4.3 --- Result and discussion --- p.46 / Chapter 4.3.1 --- Expression level of miR-122a after transfection --- p.46 / Chapter 4.3.2 --- Lentivirus-mediated miR-122a expression --- p.47 / Chapter 4.4 --- Conclusions --- p.52 / Chapter 4.5 --- References --- p.54 / Chapter Chapter 5 --- Gain-of-function study of miR-122a in HCC cells / Chapter 5.1 --- Introduction --- p.55 / Chapter 5.2 --- Experimental Section --- p.56 / Chapter 5.2.1 --- Cell culture --- p.56 / Chapter 5.2.2 --- Cell transfection --- p.56 / Chapter 5.2.3 --- Lentiviral vector transduction --- p.56 / Chapter 5.2.4 --- MTT Cell Proliferation Assay --- p.56 / Chapter 5.2.5 --- Apoptosis analysis - DAPI Staining --- p.57 / Chapter 5.2.6 --- 2-D Protein Gel Electrophoresis and MS --- p.57 / Chapter 5.2.7 --- miRNA target prediction using bioinformatic approaches --- p.59 / Chapter 5.3 --- Result and discussion --- p.59 / Chapter 5.3.1 --- Phenotypic changes of HepG2 cells caused by ectopic overexpression of miR-122a --- p.59 / Chapter 5.3.2 --- miR-122a target prediction using bioinformatic approaches --- p.62 / Chapter 5.3.3 --- Experimental validation of miR-122a targets by proteomics approach --- p.67 / Chapter 5.4 --- Conclusion --- p.70 / Chapter 5.5 --- References --- p.71 / Appendix --- p.73
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326556 |
| Date | January 2008 |
| Contributors | Diao, Shu., Chinese University of Hong Kong Graduate School. Division of Chemistry. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, vii, 83 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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