abstract / Medicine / Doctoral / Doctor of Philosophy

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.

Choi, Pui Wah. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 202-232). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts 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

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

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Two posttranslational modifications, ISGylation and phosphorylation, impact the replication of influenza A virus, a human pathogen responsible for high mortality pandemics. The ubiquitin-like ISG15 protein is induced by type 1 interferon (IFN) and is conjugated to many cellular proteins by three enzymes that are also induced by IFN. Experiments using ISG15-knockout (ISG15-/-) mice established that ISG15 and/or its conjugation inhibits the replication of influenza A virus, but inhibition was not detected in mouse embryo fibroblasts in tissue culture. The present study is focused on the effect of ISG15 and/or its conjugation on the replication of influenza A virus in human cells in tissue culture. IFN-induced antiviral activity against influenza A virus in human cells was significantly alleviated by blocking ISG15 conjugation using small interfering RNAs (siRNAs) against ISG15 conjugating enzymes. IFN-induced antiviral activity against influenza A virus gene expression and replication was reduced 10-20-fold by suppressing ISG15 conjugation. Unconjugated ISG15 does not contribute to this antiviral activity. Consequently human tissue culture cells can be used to delineate how ISG15 conjugation inhibits influenza A virus replication. SiRNA knockdowns were also used to demonstrate that other IFN-induced proteins, specifically p56, MxA and phospholipid scramblase 1, also inhibit influenza A virus gene expression in human cells. The research on phosphorylation focused on the viral NS1A protein, a multifunctional virulence factor. Although threonine phosphorylation of the NS1A protein was discovered 30 years ago, the sites of phosphorylation and its function had not been identified. A recombinant influenza A virus encoding an epitope-tagged NS1A protein was generated, enabling the purification of NS1A protein from infected cell extracts. Mass spectrometry identified phosphorylation at T49 and T215. A recombinant virus in which phosphorylation at 215 was abolished by replacing T with A is attenuated, and an apparently aberrant NS1A protein is produced. Attenuation did not occur when T was changed to E to mimic a constitutively phosphorylated state, or surprisingly when T was changed to P to mimic avian NS1A proteins. These results suggest that T215 phosphorylation in human viruses and P215 in avian viruses can support analogous functions. / text

published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

Cervical cancer is a largely preventable malignancy due to the availability of cytology screening and vaccination against the essential initiation factor of cervical carcinogenesis, human papillomavirus (HPV). However, cervical cancer remains a significant medical burden worldwide, particularly in developing countries where large scale screening or vaccination programs are not financially feasible. Molecular tests such as HPV DNA tests have the potential to improve the speed and sensitivity of cervical cancer screening but suffer from limited specificity. Additional adjunct molecular markers are therefore desirable for enhancing molecular tests. Our previous research has revealed miR-143, a microRNA downregulated in a number of cancers, could be detected in liquid based cytology samples and is significantly reduced in cervical cancer samples and cell lines. Cellular role of miR-143 and mechanism behind its downregulation remain an unknown in cervical carcinogenesis. To explore the cellular roles of miR-143 in cervical cancer, a construct expressing miR-143 was transfected into cervical cancer cell lines HeLa, SiHa and C33A. miR- 143 overexpression was verified by qPCR. The miR-143 overexpressing cell lines were used to conduct a number of cellular function assays. It has been reported that miR-143 is able to suppress cell growth in HPV-positive HeLa. We followed up the findings and revealed miR-143 overexpression in HPV-negative C33A did not suppress cell growth in an MTT cell proliferation assay. ERK5 and KRAS, two targets of miR-143, are downregulated in colon cancer and bladder cancer to suppress cell grwoth. However, mRNA level of ERK5 and KRAS were not altered in all three miR-143 overexpressed cervical cancer cell lines, suggesting that miR-143 may not target ERK5 and KRAS transcriptionally in cervical cancer. Ability of miR-143 in regulating cell differentiation was evaluated by the expression of K10, an early keratinocyte differentiation marker. K10 was upregulated only in miR-143 overexpressed HeLa and SiHa as revealed by qPCR. A parallel increase in hSkn-1a mRNA, a transcription factor of K10, was also observed specifically in the two miR-overexpressed HPV-positive cell lines. miR-143 level is inversely correlated with cytology grading and progression of cervical disease, hinting its role in mediating cell migration and invasion during cancer progression and metastasis. A reduction of cell migration as demonstrated in wound healing assay and in vitro transwell migration assay was observed exclusively in miR-143 overexpressed HeLa and SiHa. miR-143 overexpression in C33A did not introduce any effect in cell migration. A reduction of cell invasion was also observed merely in miR-143 overexpressed HeLa and SiHa as revealed in a transwell invasion assay. Apart from studying the cellular roles of miR-143 in cervical cancer, this study has also explored mechanisms behind miR-143 downregulation in cervical cancer owing to the fact that certain miR-143 mediated cellular functions were observed only in HPV-positive cervical cancer cell lines. We hypothesized that HPV E6 and E7 oncoprotein may downregulate miR-143 in cervical cancer. The hypothesis was supported by our findings where normal cervical epithelial cell line immortalized by E6 and E7 had an undetectable level of endogenous miR-143 level. The same primary cells immortalized by shp16-hTERT expressed residual amounts of miR-143 as revealed by qPCR. Owing to the low miR-143 expression in shp16-hTERTimmortalized normal cervical epithelial cell line, downregulation of miR-143 in cervical cancer cell lines may also be contributed to hTERT overexpression and p16 silencing. Overall, miR-143 plays an important role in suppressing cell proliferation, enhancing keratinocyte differentiation marker expression, reducing migration and invasion in HPV-positive cervical cancer. Downregulation of miR-143 level may be an effect as manifested by E6 and E7 in HPV-positive cervical cancer. Differential cellular effects in miR-143 overexpressed HPV-positive and HPV-negative cervical cancer cell lines suggest that HPV oncoprotein mediates miR-143 cellular functions. / published_or_final_version / Pathology / Master / Master of Medical Sciences

Multiple myeloma (MM) is an incurable haematological malignancy. It is characterized clinically by an asymptomatic precursor stage, known as monoclonal gammopathy of undetermined significance (MGUS), which will transform into symptomatic MM at a rate of 1% per year. Gene promoter hypermethylation by catalytic conversion of cytosine into 5?methylcytosine at promoter?associated CpG island is an alternative mechanism of gene inactivation. MicroRNA (miRNA) is a class of short, single?stranded, non?coding RNA molecules, which will repress the expression of target protein by sequence?specific binding to the three prime untranslated region of the corresponding messenger RNA. In carcinogenesis, miRNA can be either oncogenic when tumour suppressor genes are targeted, or tumour suppressive when oncogenes are targeted. Despite reports of hypermethylation of multiple protein?coding tumour suppressor genes, little is known about DNA methylation of non?coding tumour suppressor miRNA genes in MM. This thesis aimed to investigate the role of promoter hypermethylation of tumour suppressor miRNA genes in MM using a candidate miRNA approach. Moreover, the prognostic significance of tumour suppressor miRNA hypermethylation was studied in a uniformly?treated cohort of MM patients. The role of DNA methylation at the promoter of miR?203, miR?34a, miR?34b/c, miR?124?1, miR?129?2 and miR?224 were studied in MM. The tumour suppressor role of miR?34b/c, miR?124?1, miR?203 and miR?224 were demonstrated in human myeloma cell lines (HMCLs). In particular, restoration of miR?203 in MM cells was shown to inhibit cellular proliferation via targeting and hence direct downregulation of a proto?oncogene, cyclic AMP responsive element binding protein. There are several observations in primary MM samples. First, there was frequent methylation of miR?129?2, miR?203 and miR?224 but infrequent methylation of miR?34a, miR?34b/c and miR?124?1 in MM at diagnosis. Second, tumour?specific hypermethylation of each of the miR?203 and miR?224 promoters was detected at comparable frequencies in MGUS, diagnostic and relapsed/progressed MM, and hence implicated as an early event in myelomagenesis. Thirdly, miR?129?2 methylation was more frequent in diagnostic MM than MGUS, and hence implicated in MGUS progression to MM. On the other hand, despite rare miR?34b/c methylation at diagnosis, miR?34b/c methylation was frequent at relapse/progression, thereby implicating miR?34b/c methylation in MM relapse/progression. Fourthly, despite frequent miR?124?1 methylation in HMCLs, miR?124?1 methylation was rare in both diagnostic and relapsed MM marrow samples, suggesting that miR?124?1 methylation was acquired during in vitro cell culture. Finally, the prognostic significance of methylation of a panel of tumour suppressor miRNAs was studied in a uniformly?treated cohort of MM patients, which revealed that miR?224 hypermethylation as an independent favourable prognostic factor for survival. In conclusion, hypermethylation of tumour suppressor miRNAs is implicated in the pathogenesis (miR?203, miR?129?2, miR?224), progression (miR?34b/c), and prognostification (miR?224) of MM. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy