Nasopharyngeal carcinoma (NPC) is different from other head and neck squamous cell carcinomas and is closely related with Epstein-Barr virus infection. It is endemic in southern China and Southeast Asia, affecting between 20 and 30 per 100,000 populations. According to the World Health Organization (WHO) histological classification, there are three subtypes of NPC: WHO type 1 NPC is keratinizing squamous cell carcinoma; type 2 NPC is differentiated non-keratinizing carcinoma; type 3 NPC is undifferentiated non-keratinizing carcinoma. In southern China including Hong Kong, type 3 NPC (undifferentiated NPC) is dominant and constitutes over 90% of the total NPC. MicroRNA-138 (miR-138) is a small non-coding RNA which has been reported to be highly expressed in undifferentiated NPC. We hereby evaluated whether the miR-138 level could be used to differentiate NPC patients from the normal individuals and examine the potential oncogenic role in undifferentiated NPC cell line. To validate the hypothesis that miR-138 was an oncogenic microRNA, which is overexpressed in undifferentiated NPC patients, we first examined its expression level in nasopharyngeal tissues and peripheral blood. In our cohort, cancer tissues samples were collected from 42 primary NPC and 29 recurrent NPC patients. To evaluate the expression level in the cancer tissues, the miR-138 level was quantified by real-time quantitative polymerase chain reaction. For primary NPC, the expression level was compared with 29 normal nasopharyngeal epithelia. For recurrent NPC, the microRNA level was compared with the paired normal mucosa counterparts obtained from the same patients. In addition, plasma samples were also collected from 22 primary NPC, 21 recurrent NPC and 17 normal individuals. Our data suggested that there was no difference in the miR-138 expression level in primary NPC tissue and normal nasopharyngeal tissue from control. There was no difference in the circulating miR-138 levels in the primary NPC, recurrent NPC and normal control groups. The circulating miR-138 could not be used to differentiate NPC patients from the normal individuals. Further functional analysis on the undifferentiated NPC cell line HONE1 suggested that miR-138 overexpression could enhance NPC cell proliferation, migration and invasion in comparison with the mock control. With the use of high-throughput gene expression arrays, we observed that multiple cancer-related pathways were affected in miR-138 overexpressed NPC cells. Staining with Acridine orange (AO) and phosphorylated H2AX (γH2AX) showed that miR-138 overexpression is associated with an enhanced response to radiation. Our results are concordant with other similar studies suggested that miR-138 is an oncogenic microRNA which play an important part in the undifferentiated NPC tumorigenesis. Further studies, based on larger sample size, are warranted to explore the clinical use of this small RNA in diagnosis, prognosis and management of undifferentiated NPC. / published_or_final_version / Surgery / Master / Master of Philosophy

The groundbreaking use of transcription factors (Oct4, Sox2, Klf4, c-Myc) in reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) provides novel ways in regenerative medicine and disease modeling. The reprogramming process is a stepwise process involving global epigenetic remodeling. In recent years, small molecules like DNA methyltransferase inhibitor that alter the epigenetic status of cells were shown to enhance the reprogramming efficiency. It was postulated that chromatin modifying enzymes played an important role during the reprogramming process, and microRNAs (miRNAs) were the upstream regulators. The objectives of this study involve the identification of potential miRNAs regulating the expression of chromatin modifying enzymes and the study of their roles during reprogramming. Primary mouse embryonic fibroblasts (1o MEFs) were used for the establishment of a reprogramming system, where the delivery of transcription factors Oct4, Sox2, klf4 and cMyc was mediated by lentivirus. Another established secondary MEFs (2o MEFs) reprogramming system was also included in the study. Mouse iPSCs (miPSCs) derived from both systems were shown to express pluripotent markers. In-silico analysis predicted a set of miRNAs (miR-101, miR-135a, miR-148a and miR-148b) commonly targeted the chromatin modifying enzymes in mouse genome. Among them, miR-101 and miR-135a overexpression were found to inhibit the reprogramming efficiency significantly in both 1o and 2o MEFs. Conversely, the inhibition of miR-135a but not miR-101 expression significantly enhanced the reprogramming efficiency in both systems. In this study, it was postulated that miR-101 regulated enhancer of zeste homolog 2 (Ezh2) during reprogramming. Ezh2 was confirmed to be negatively regulated by miR-101 at protein level. The expression of Ezh2 was high in mouse embryonic stem cells (mESCs) but time dependently depressed during mESC differentiation, while its expression was increased during reprogramming of MEFs. Ezh2 expression was found to negatively correlate with miR-101 expression in these conditions. In addition, the knockdown of Ezh2 mimicked the inhibitory effect of miR-101 overexpression on reprogramming efficiency. The inhibitory role of miR-135a on reprogramming was linked to its potential target, Sirtuin 1 (Sirt1). Sirt1 was negatively regulated by miR-135a. The expression of miR-135a was upregulated upon mESC differentiation and decreased during reprogramming. Together with the previous finding in this laboratory, miR-135a expression was negatively correlated with Sirt1. Furthermore, miR-135a inhibition increased the proliferation rate of MEFs. More importantly, miPSCs reprogrammed from miR-135a knockdown MEFs maintained the pluripotent state. To further analyze the pluripotency of the miPSCs, the tetraploid complementation assay was established. Preliminary studies were performed to optimize the conditions for electrofusion. Although single electrofusion with a lower field strength (1000V/cm) resulted in lower fusion rate, the development of the mESC aggregated embryo was the best when compared to higher field strength and those with double electrofusion. Lastly, the mESCs aggregated into tetraploid embryo were mainly localize in the inner cell mass of the embryo. In conclusion, negative correlations were found between miR-101/Ezh2, and miR-135a/Sirt1 during somatic cell reprogramming. The identification of small molecules in reprogramming helps to understand the molecular mechanisms of reprogramming. / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

Background: Esophageal squamous cell carcinoma (ESCC) has been increasing in incidence, but knowledge of the genetic basis of this disease remains limited. In general, esophageal carcinoma can be divided into two main types: Esophageal Squamous Cell Carcinoma (ESCC) and Esophageal Adenocarcinoma (EAC). The pathogenesis of esophageal carcinoma still remains unclear, although some risk factors like chronic irritation, or chronic inflammation which may be caused by diseases such as gastroesophageal reflux disease (GERD) or unhealthy lifestyles like smoking have been proved to be related to the carcinogenic process. Diagnosis and treatment for this kind of cancer have continue to develop and evolve, but the 5-year overall survival rate is still relatively low. Therefore, it is clinically important to identify any potential genetic changes which may help us to discover some useful biomarker targets for the further development of more direct and harmless targeted therapy for our esophageal cancer patients. Objectives: In this study, I aimed to identify some potential oncogenic microRNA (miRNA) and to study their clinical meaning in ESCC patients. Methods: Microarray was applied to identify differentially expressed miRNAs in ESCC tumour tissue, compared with corresponding adjacent non-tumour esophageal tissue. One candidate oncogenic miRNA, miR-665, was investigated in the present study. After testing the expression level of miR-665 in ESCC cell lines and patients’ samples with RT-PCR, miR-665 stably expressing cells was established using two ESCC cell lines (KYSE30 and KYSE510). Functional characterization was then conducted using in vitro and in vivo assays to examine the effect of miR-665 towards the development of ESCC. Bioinformatic software such as Target Scan was used to generate a list of predicted target genes that may be modulated by miR-665. Results: The high expression of miR-665 has been confirmed in ESCC tissues and cell lines, showing the potential carcinogenic function of miR-665. Ectopic expression of miR-665 also demonstrates its ability to enhance tumour growth and invasion in vitro and in vivo. Bioinformatic analysis of miR-665 predicted targets showed putative binding sites for miR-665 within the 3’UTR region of NLK. Conclusions: This study has identified a novel miRNA and a related gene which might play an important role in the pathogenesis of ESCC, affecting the cancer process and tumour growth. This may help to find potential new biomarker for the future improvement and development of new treatment of ESCC patients. / published_or_final_version / Clinical Oncology / Master / Master of Philosophy

Hepatocellular carcinoma (HCC) is the fifth most prevalent human malignancy and the third leading cause of cancer deaths in the world. MicroRNAs (miRNA) are conserved, small noncoding RNA molecules that regulate gene expression of protein-coding gene posttranscriptionally. Dysregulation of miRNA is implicated in many human malignancies including HCC, yet little is known regarding the regulatory mechanisms of these small noncoding RNAs. Hypoxia is a prevalent! tumor microenvironment in HCC because of its rapid growth often to large size and plays a key role in modulating tumor aggressiveness. In the present study, we investigated the effects of hypoxia on microRNA expression in human HCCs, identified and characterized hypoxia-inducible microRNAs that are important for the development of aggressive phenotypes. To initialize the study, we examined the miRNA expression profiles with TaqMan human microRNA Low-Density Array and identified a panel of microRNAs differentially expressed in HCC cells under hypoxic conditions. We observed that miR-210 was consistently upregulated by hypoxia in a total of 7 different HCC cell lines, via a HIF1α-dependent mechanism. In human HCCs, miRL210 overexpression significantly correlated with poorer overall and disease-free survival of patients, as well as aggressive pathologic features, including advanced tumor stages of HCC and the presence of venous invasion. These findings established miRL210 as a surrogate marker of aggressive HCC with high metastatic potential. In most human malignancies, cancer metastasis contributes to about 90% of cancer-related mortality. Given the correlation of miR-210 levels with poorer patient survival and aggressive clinical features of HCC, we then characterized the metastatic role of miRL210 by functional assays in the second part of the study. The findings from in vitro and in vivo experiments using both gain- and loss-of-function approaches led us to conclude that the hypoxic induction of miRL210 enhanced metastatic potential of HCC cells. The pro-metastatic effect of miRL210 was attributed, at least partly, to the downregulation of TIMP2 by hypoxia, through a feedback loop circuit consisting of HIF1α, miRL210, and HIF3α. The impact of miR-210 on HCC metastasis was not the only scope of this study since hypoxia has long been recognized as a major obstacle in chemotherapy. Given that activation of the HIF1α-miR-210 axis was frequently observed in hypoxic HCC cells, in the last part of the study we also investigated whether hypoxic induction of miRL210 promoted cell survival against cytotoxic treatments, including cisplatin and 5-flurouracil. Here, we demonstrated that induction of HIF1α-miR-210 axis conferred chemoresistance to HCC cells under hypoxic conditions, and inhibition of miR-210 re-sensitized HCC cells to these cytotoxic drugs. Mechanistically, we also revealed that RAD52 was a direct functional target of miRL210 that linked hypoxia to chemoresistance in HCC cells. The overall findings of this study have enriched our understanding of miR-210 as a mediator of hypoxic responses in HCC, in particular metastasis and chemoresistance. We have highlighted the clinical significance of this microRNA by showing that miR-210 can serve not only as a prognostic marker for HCC progression, but also as a mediator for the hypoxic tumor microenvironment to modulate tumor aggressiveness. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Introduction Globesity, the worldwide obesity epidemic, represents a major threat and public health burden. An uncontrolled expansion of the adipose tissue followed by a chronic low-grade inflammation leads to the dysfunction of the adipose organ resulting in obesity and its associated metabolic complications. Uncovering the mechanisms of adipogenesis, the development of adipocytes, therefore strikes as a key strategy in combating the disease. MicroRNAs (miRs), a class of small non-coding RNAs, have emerged in recent years as crucial modulators of diverse biological processes such as cell proliferation, differentiation, and signal transduction emphasizing their large potential as targets. Numerous miRs have been associated with the adipose tissue and metabolism and their dysregulation has repeatedly been linked to diseases including diabetes and obesity. This study aimed to investigate the role of miR-34a, an obesity-related miR, in the regulation of pre-adipocyte differentiation. Materials and Methods Mouse 3T3-L1 pre-adipocytes were employed as an in vitro system to study adipogenesis. Oil Red O staining served to evaluate the degree of adipogenesis and the over-expression of miR-34a in adipocytes was achieved by a lentiviral system. MiR and messenger RNA (mRNA) levels were analysed using TaqMan and SYBR Green-based quantitative real time PCR (qPCR) respectively. Results The expression of miR-34a was substantially down-regulated upon treatment of differentiation medium for two days and remained significantly low during the differentiation period compared with undifferentiated pre-adipocytes. Lentivirus-mediated over-expression of miR-34a successfully up-regulated miR-34a. Higher levels of miR-34a in turn mitigated adipogenesis as evidenced by blunted Oil Red O staining. This observation was found to be in good agreement with the qPCR analysis, which showed a down-regulation of several key adipogenic markers. Conclusion The down-regulation of miR-34a is required during pre-adipocyte differentiation for the efficient proceedings of the adipogenic programme. Further investigation is needed to evaluate the potential therapeutic implication of miR-34a-based treatment in managing obesity. / published_or_final_version / Medicine / Master / Master of Medical Sciences

Choriocarcinoma is a malignant form of gestational trophoblastic disease arising from the trophoblastic epithelium. It is characterized by the presence of a mixed population of mononuclear cytotrophoblasts and multinucleated syncytotrophoblasts surrounded by hemorrhage and necrosis. Clinically, it is difficult to distinguish postmolar choriocarcinoma from an invasive mole. They also share similar histopathological features and are only distinguishable from invasive moles by the absence of chorionic villi. Since choriocarcinoma is an aggressive tumor with a high tendency to metastasize, it is better to have a definitive diagnosis to detect the disease at an earlier stage in order to tackle the problem before it becomes too advanced. It is thus necessary to investigate new potential markers which could help in making diagnosis at the early stage of the disease. MicroRNAs are recognized as a new class of non-coding RNAs that regulate gene expressions post-transcriptionally through translational repression or degradation of the target messenger RNAs. They are involved in almost every biological process, including cell proliferation, differentiation as well as apoptosis. MiR-125 is one of the most widely investigated microRNAs in recent years, particularly in cancers. It is a highly conserved sequence that expresses ubiquitously in multiple human organs in a tissue-specific manner. The deregulation of miR-125 was commonly found in various types of cancers. Depending on the target messenger RNAs, miR-125 exerts either tumor suppressive or oncogenic effects. There are three homologues of miR-125, including miR-125a, miR-125b-1 and miR125b-2. Since all three homologues have very similar sequence and have the same seed region, they have common mRNA targets and similar functions but may express differentially in different tissues. Factor that binds to inducer of short transcript-1(FBI-1) is a transcription factor that is involved in cell cycle arrest and terminal differentiation in different tissues. The deregulation of FBI-1 was associated with oncogenesis and the overexpression of FBI-1 was frequently demonstrated in multiple human cancers. However, the connection between miR-125 and FBI-1 in choriocarcinoma has not been reported. In this study, an inverse relationship between miR-125, including both miR-125a and miR-125b, and FBI-1 was demonstrated. Higher expression levels of miR-125a and miR-125b were demonstrated in the first-trimester extravillous trophoblasts,TEV-1, than in the JAR and JEG-3 choriocarcinoma cells, whereas the protein and mRNA expression levels of FBI-1 were significantly higher in JAR and JEG-3 cells than in TEV-1 cells. Moreover, the overexpression of miR-125 down-regulated the FBI-1 expression level in both JAR and JEG-3 cells, suggesting that miR-125 may regulate FBI-1 through a direct interaction. By treating JEG-3 cells with a histone deacetylase inhibitor, trichostatin A (TSA), the expression levelsof miR-125a and miR-125b were up-regulated while the FBI-1 was down-regulated, suggesting the possible transcriptional silencing effect on miR-125 through histone deacetylation. Altogether, miR-125 affects FBI-1 expression and may serve as a new marker to differentiate malignant tumors from the benign GTD as well as a therapeutic target. / published_or_final_version / Pathology / Master / Master of Medical Sciences

Epithelial ovarian cancer is a common female malignancy with a relatively high mortality rate worldwide. This may due to a lack of efficient diagnostic methods at early stage and worsen by complications caused by metastasis at advanced stage. For successful metastasis, cancer cells detached from the original growing sites have to survive in the body circulation before conquering a distant location within the body. Resistance to anoikis (apoptosis induced without appropriate extracellular matrix) is therefore utmost important during metastatic spreading. In addition, a pre-metastatic niche remodeled by cancer cells is also a pre-requisite for metastatic colonization. Emerging evidence has suggested the dysregulation of miRNAs is associated with different aspects of tumorigenesis. However, the specific roles of miRNAs in anoikis resistance and in remodeling of distant niche remain unknown thus far. This study attempted to investigate the functional roles of miR-141, in particularly anoikis resistance of ovarian cancer cells and the reprogramming of stromal cells. The miR-200 family is frequently upregulated and associated with human cancer metastasis. In this study, by cDNA array profiling together with biochemical and functional studies, miR-141, a member of miR-200 family, was identified as an oncomiR enhancing cell viability in low serum medium and anoikis resistance. Moreover, enforced expression of miR-141 led to bigger tumor sizes and promoted metastatic colonization in mouse models. Further studies demonstrated miR-141 directly targets tumor suppressive KLF12 in ovarian cancer cells, depletion of KLF12 could mimic function of miR-141. Clinical study revealed the upregulated miR-141 was significantly correlated with the downregulated KLF12, serous subtype, advanced and distant metastatic ovarian cancer. Furthermore, Genechip profiling, Human Apoptosis Array and Luciferase reporter assay revealed the upregulated miR-141 and downregulated KLF12 enhanced anoikis resistance via elevation of survivin which protect cells against intrinsic apoptotic activity. On the other aspect, miR-141 was found to be a secretary miRNA and commonly detected in the serum of ovarian cancer patients. The upregulated miR-141 expression was also correlated to levels of common cancer biomarker CA125. Importantly, the serum miR-141 level was significantly correlated with the tumor burden of patients during treatments, indicating it could be used as a non-invasive biomarker for ovarian cancers. Finally, based on miR-141 as tumor-secreted and circulated miRNA, a series of functional studies demonstrated that miR-141 could be transferred to hFF-1 fibroblast cells. Intriguingly, ovarian cancer cells cultured in miR-141-fibroblast culturing medium showed a remarkable increase of cell migration, suggesting that the remodeled-miR-141 fibroblast cells can secrete stimulating factors and promote ovarian cancer cells aggressiveness. This is the first study showing miR-141 could reprogram fibroblast cells to be a niche for ovarian cancer cell dissemination and metastatic progression. However, further investigations for verifying such functions are warranted. In conclusion, this study provides strong evidence that miR-141 is oncoMir enhancing ovarian cancer cell plasticity in metastasis e.g. anoikis resistance. Moreover, the finding of secretary form miR-141 not only gives the feasibility to be a potential biomarker for detecting ovarian cancer but also shows a possible mechanism of how miRNAs reprogram the distant niche for metastatic colonization. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Computer Science and Information Systems / Master / Master of Philosophy

published_or_final_version / abstract / toc / Biochemistry / Master / Master of Philosophy

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