In conclusion, downregulation of miR-124 and miR-383 is a frequent event in MB. Restoration of miR-124 and miR-383 inhibited cell growth and cell cycle progression in MB, suggesting these miRNAs harbor growth suppressor function. In addition, this study demonstrates SLC16A1 and PRDX3 are the direct targets of miR-124 and miR-383, respectively. Together, these data shed new light on miR-124 and miR-383 in MB pathogenesis, and suggest that miR-124/SLC16Al and miR-383/PRDX3 pathways are potential therapeutic targets for treatment of MB. / Medulloblastoma (MB) is an invasive embryonal tumor of the cerebellum, accounting for ∼20% of all primary pediatric brain tumors. The overall survival rate is 60--70% in standard-risk MB patients, but merely ∼30% in high-risk group. Patients who survive often suffer from long-term neurologic and cognitive deficits. New therapy is needed to reduce the mortality rate and to improve the quality of life of survivors. Understanding the molecular pathogenesis of MB is critical to the development of efficacious therapeutic treatment. / MicroRNAs (miRNAs) are short non-protein-coding RNAs that function in diverse biological processes through negative regulation on gene expression at the post-transcriptional level. Accumulative evidence indicates that miRNAs play an important role in the development of human cancers, with their deregulation resulting in altered activity of downstream tumor suppressors, oncogenes and other signaling molecules. / The aim of my project is to identify and characterize deregulated miRNAs located on chromosome 8p in MB. Our group has previously identified a minimally deleted region on 8p22-23.1 and partial or interstitial deletions at 8p22-23.2 in MBs. Despite extensive investigation, no promising candidate genes were identified in these. I questioned if miRNAs (miR-124, miR-383 and miR-320) were the targets on chromosome 8p. Quantitative expression analysis of 29 MBs revealed that miR-124 and miR-383 were downregulated in 72% and 79% of tumors, respectively, compared to normal cerebella. In contrast miR-320 expression was variable. Ectopic expression of miR-124 and miR-383 in MB cell lines (DAOY and ONS-76) showed significant growth inhibition. Cell cycle profiling revealed miR-124 and miR-383 inhibited cell cycle progression and induced apoptosis. These results suggest that miR-124 and miR-383 are potential growth suppressors. / To identify gene targets of miR-124, computational analysis was carried out. Twelve candidate genes predicted as miR124 target were selected for analysis. One candidate gene, SLC16A1, showed downregulation at transcript and protein levels after miR-124 transfection. Luciferase reporter assay demonstrated that miR-124 interacted at the 3' untranslated region of SLC16A1. These results suggest that miR-124 negatively regulates SLC16A1. Expression analysis further revealed that overexpression of SLC16A1 was common in MBs. / To identify miR-383 targets, global gene expression analysis and computational approach were applied. Two genes (PRDX3 and RBMS1 ) showed downregulation upon miR-383 transfection. Reporter assay confirmed that miR-383 interacted at 3' untranslated regions of these genes, suggesting that PRDX3 and RBMS1 are targets ofmiR-383. / Li, Ka Wai Kay. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 236-293). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

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

Medulloblastoma (MB) and supratentorial primitive neuroectodermal tumor (stPNET) are pediatric embryonic brain tumors, which arise in a brain that is in the process of growth and development. They differ significantly from adult lesions and may involve unique genetic and epigenetic factors. However, the pathogenesis of these tumors is still elusive. My project consisted of four parts, investigating major genetic and epigenetic alterations of these tumors. / Multiple genetic studies have shown high frequency of loss (30--60%) on chromosome 8p in MBs. Microcell-mediated transfer of chromosome 8 suppressed tumorigenesis or the proliferation of colon and breast cancer cell, indicating that chromosome 8p is likely to include several TSGs in human cancers. In previous studies from our laboratory, results showed the frequency of loss on chromosome 8p is also rather high (66.7%). An overlapping HD region was identified in a 1.8cM interval on 8p22-23.1, between markers D8S520 and D8S1130, in two MBs (Yin et al., 2002), indicating that several candidate TSGs are located within or near this region. PinX1 on 8p23.1, a potential inhibitor of telomerase, is most likely the candidate TSG in MBs due to its location and function. To evaluate the genetic alterations of PinX1 and to investigate its role in MBs, the first part of my study is to perform mutation analysis in a series of 52 primary MBs, 3 MB cell lines and 4 primary stPNETs. Transcript expression of PinX1 was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) in microdissected tumors and normal cerebellum. Using the telomeric repeat amplification protocol (TRAP) assay, 19 MBs, 2 stPNETs and all 3 MB cell lines were analyzed for telomerase activity. No somatic point mutations and loss of expression of PinX1 were detected in our series, suggesting that PinX1 is not the target gene on 8p23.1 in MBs. Although we did not find a significant association between PinX1 expression and telomerase activity, the presence of telomerase activity in 16 of 22 MBs and 1 of 2 stPNETs indicate that telomerase activation is associated with the development of this malignant disease. Our study represents the largest series of MB examined by telomerase repeat amplification protocol (TRAP) assay. (Abstract shortened by UMI.) / Chang Qing. / "April 2005." / Adviser: Ho-Keung Ng. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0191. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 201-228). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.