published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

在不同的模型體系和疾病microRNA-199a (miR-199a)的功能和基因表達都不盡相同而且相當複雜。它在人基因組中有個表達位點，分別是位於19 號染色體的miR-199a-1 和1 號染色體的miR-199a-2。這個位點可以產生相同的miRNA (miR-199a-3p 和miR-199a-5p)。造成miR-199a不同表達和功能的原因尚未清楚。但是，很有可能是由於個位點不同的調節機制和miR-199a 在不同的細胞和組織中可變的作用对象。在睾丸生殖細胞腫瘤(簡稱睾丸癌)和膠質母細胞瘤(簡稱膠質瘤)中研究miR-199a 的啟動子甲基化發現，在睾丸癌中miR-199a-1 和-2 共同的啟動子過甲基化導致miR-199a 表達低。而在膠質瘤中，只有miR-199a-2，而不是miR-199a-1 的啟動子低甲基化與miR-199a 的高表達相關。除啟動子甲基化，在幹細胞分化過程中升高表達的miR-199a 被證實是通過轉因子Twist1 的調控；循环通Twist1-miR-199a-5p-HIF1α控制miR-199a-5p 促進成骨分化的作用。miR-199a 的功能是通過其下游目標 (靶基因) 而成的。因此, 目標分子的功能控制miR-199a 在同的模型體系和疾病中的作用。我應用基因組學和蛋白組學的方法尋找miR-199a-5p 在睾丸癌中的下游目標(靶基因)。MAFB 被指出和證明是miR-199a-5p 在睾丸癌中的靶基因，而miR-199a 抗腫瘤細胞增殖活性的作用是通過MAFB 發揮的。通過研究miR-199a 在用系統中的調控機制和作用目標，我希望用miR-199a 作為模型明miRNA 生物學的複雜多樣性。 / microRNA-199a (miR-199a) has been shown to have diverse biological functions and behave quite differently in different physiological systems and diseases. It is encoded by two loci in the human genome, miR-199a-1 in chromosome 19 and miR-199a-2 in chromosome 1. Both loci give rise to the same miRNAs (miR-199a-5p and miR-199a-3p). The underlying mechanism responsible for the diverse action of the miRNA is not clear. However, it is likely contributed by differential regulation of the two genomic loci and variable targets of the miRNA in different cells and tissues. Studies on the promoter methylation of miR-199a in testicular germ cell tumors (TGCTs) and glioblastomas (gliomas) demonstrated that hypermethylation of both loci of miR- 199a resulted in its reduced expression in TGCTs, while hypomethylation of miR-199a-2 but not -1 in gliomas might lead to its elevated expression. In addition to DNA methylation, the functions of transcription factors in controlling the expression of miR-199a through a Twist1- miR-199a-5p-HIF1α cyclic pathway were demonstrated to play significant roles during me nchymal stem cell differentiation. The functions of miR-199a are mediated by the actions of its downstream targets. Both genomic and proteomic approaches were applied to study the targets of miR-199a-5p in TGCTs. A putative target of miRNA-199a-5p, MAFB, was identified and confirmed to be responsible for the anti-tumor proliferation activity of the microRNA. By studying the mechanisms that control the expressions of miR-199a and its various downstream targets in different systems, it is hoped that miR-199a could be used as a model to illustrate the complexity of miRNA biology. / Detailed summary in vernacular field only. / Gu, Shen. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 144-163). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / Abstract --- p.I / 摘要 --- p.II / Acknowledgements --- p.III / Abbreviations --- p.VIII / Chapter 1 --- p.1 / Chapter 1.1 --- miRNAs - small yet powerful molecules --- p.2 / Chapter 1.1.1 --- Biogenesis and processing of miRNA --- p.2 / Chapter 1.1.2 --- Detection of miRNA expression --- p.7 / Chapter 1.1.3 --- Identification of putative miRNA targets --- p.11 / Chapter 1.2 --- miR-199a - an example revealing the complexity of the miRNA world --- p.19 / Chapter 1.2.1 --- miR-199a - standing out among thousands --- p.19 / Chapter 1.2.2 --- Regulation of miR-199a expression --- p.20 / Chapter 1.2.3 --- miR-199a in carcinogenesis --- p.24 / Chapter 1.2.4 --- miR-199a in cardiogenesis --- p.33 / Chapter 1.2.5 --- miR-199a in stem cell differentiation and embryo development --- p.34 / Chapter 1.2.6 --- Other functions of miR-199a --- p.35 / Chapter 1.3 --- Project overview --- p.38 / Chapter 2 --- p.39 / Chapter 2.1 --- Introduction --- p.40 / Chapter 2.2 --- Materials and methods --- p.42 / Chapter 2.3 --- Results --- p.49 / Chapter 2.3.1 --- Dysregulation of miR-199a in tumors were controlled by DNA methylation --- p.49 / Chapter 2.3.2 --- Transcriptome changes upon miR-199a-5p in TGCTs --- p.58 / Chapter 2.3.3 --- Identification of MAFB as a putative target of miR-199a-5p in TGCTs --- p.63 / Chapter 2.3.4 --- MAFB is highly expressed in malignant testicular tumor and negatively correlated with miR-199a-5p --- p.67 / Chapter 2.3.5 --- MAFB knockdown suppresses tumor cell growth in vitro --- p.69 / Chapter 2.4 --- Discussion --- p.70 / Chapter 3 --- p.75 / Chapter 3.1 --- Introduction --- p.76 / Chapter 3.2 --- Materials and methods --- p.81 / Chapter 3.3 --- Results --- p.86 / Chapter 3.3.1 --- Expression of miR-199a during MSC osteogenesis --- p.86 / Chapter 3.3.2 --- Functions of miR-199a on osteogenesis of hMSCs --- p.90 / Chapter 3.3.3 --- Involvement of HIF1α, Twist1 and miR-199a-5p during osteogenesis of hMSC at early stage and late stage of differentiation --- p.94 / Chapter 3.3.4 --- Up-regulation of miR-199a-5p was related to hypoxia enhanced osteogenesis at early stage --- p.99 / Chapter 3.3.5 --- miR-199a-5p inhibited HIF1α-Twist1 pathway to increase osteogenesis at late stage --- p.103 / Chapter 3.4 --- Discussion --- p.107 / Chapter 4 --- p.111 / Chapter 4.1 --- Overview of the project --- p.112 / Chapter 4.2 --- Summary and conclusion --- p.115 / Chapter 4.3 --- Future work --- p.116 / Chapter Supplementary Materials --- p.117 / Chapter Supplementary Table 2.1 --- p.118 / Chapter Supplementary Table 2.2 --- p.129 / Chapter Supplementary Table 2.3 --- p.142 / References --- p.144

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 73-82) Also available in print.

Thesis (M. Med. Sc.)--University of Hong Kong, 2008. / Includes bibliographical references (p. 89-107)

Thesis (M.S.)--Montana State University--Bozeman, 2005. / Typescript. Chairperson, Graduate Committee: A. Michael Babcock. Includes bibliographical references (leaves 40-45).

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

The Cantell strain of Sendai virus (SeV-C) represents a typical laboratory attenuated virus which is less virulent and able to induce large amount of interferon in infected cells. This strain has widely been used in the laboratory for immunological studies due to its extraordinary ability to stimulate type I interferon production. Nevertheless, the underlying mechanism by which SeV-C is sensed by immune receptors as an invading microorganism is still largely unknown. Meanwhile, during the course of infection, Sendai virus is known to generate substantial amount of non-infectious viral particles consisting of defective interfering RNAs (DI RNAs) from replication errors. It was known that one major form of DI RNAs generated by some negative stranded RNA viruses may adopt a stem-loop panhandle structure with a relatively long double stranded region. Therefore, we hypothesized that some SeV-C DI RNAs may bear structures similar to intermediate-length dsRNA recognized by cytosolic immune receptor RIG-I, thus triggering interferon production. In this study, three DI RNAs were successfully isolated from SeV-C infected cells. Particularly, one of them designated T4 was found to contain a double stranded region of 93 base pair, and it was capable of stimulating interferon β production when transfected to reporter cells. The immunostimulatory activity of T4 alone was as potent as that of SeV-C, suggesting that T4 would be the major component in attenuated virus SeV-C that activates type I interferon production. Furthermore, cellular dsRNA binding protein PACT was shown to play a role in T4 recognition by RIG-I. T4 binds to PACT and potently stimulates PACT-induced activation of RIG-I. The identification and characterization of T4 reveals the major immunostimulatory component in SeV-C. Our work defines a RIG-I agonist naturally produced during the course of viral infection and provides a new mechanism to explain virus attenuation. We also demonstrate the role of PACT in immune sensing of a viral RNA. In addition, our findings also provide new strategies for the design and development of vaccines, vaccine adjuvants and other immunostimulatory agents. / published_or_final_version / Biochemistry / Master / Master of Philosophy

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