Merkel cell carcinoma (MCC) is a rare but highly aggressive neuroendocrine cutaneous cancer whose molecular biology is poorly characterized. Our broad research objective is to identify microRNAs (miRNA) that are biologically and/or clinically important in MCC. While attempting to establish an MCC-specific miRNA signature, we observed that microRNA-375 was the most highly expressed miRNA in primary MCC tumours relative to normal skin – an observation that I propose reflects miR-375’s specific association with neuroendocrine (NE) and secretory subpopulations within normal tissues. Here, I report that miR-375 is strikingly elevated in a range of NE tumour types compared with tissue-matched cancers of non-neuroendocrine origin. Furthermore, I show that miR-375 is expressed abundantly in a subset of MCC cell lines that possess the biochemical and immunohistochemical characteristics of NE cells, but is silenced in cell lines that fail to retain these markers. I demonstrate that the enforced expression of miR-375 induces a NE gene expression signature – a phenomenon that is mechanistically driven by the post-transcriptional repression of multiple Notch pathway components by miR-375. This work identifies the Notch pathway as a novel mechanistic link between the association of miR-375 and a NE cell fate, provides new insights into the cellular ancestry of MCC, and suggests that miR-375 could facilitate clinicopathological diagnosis of MCC and other NE tumours as a novel biomarker. miR-375 is silenced in “variant” MCC cell lines, and inversely correlates with cell doubling time and overall aggressiveness. Therefore, despite its high expression in most MCC tumours, I propose that miR-375 is an endogenous tumour suppressor. I show that the enforced expression of miR-375 inhibits cell viability, impairs cell migration and invasion, can oppose survival under stress, and represses the AKT pro-survival signaling pathway. Only siRNA-mediated inhibition of Notch2 and Recombination signal binding protein for immunoglobulin kappa J region (RBPJ) phenocopied the effects of miR-375 overexpression. Because variant (miR-375low) cell lines originate from more aggressive tumours in both MCC and small cell lung carcinoma, I postulate that miR-375 silencing occurs in a subset of MCC patients and might predispose them to a highly virulent clinical course through the disinhibition of Notch signaling. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2013-08-15 09:59:09.832

MicroRNAs (miRNAs) are short, non-coding RNA sequences which regulate gene expression. Regulation is mediated primarily through binding to complementary sites in their un-translated regions which leads to mRNA degradation or translational repression. Hypoxia is a known feature of many tumours, and increased hypoxia is associated with poor prognosis. Hypoxia leads to the up-regulation of many genes involved in a variety of functions including angiogenesis, a shift to glycolytic metabolism, and cell proliferation. This is mediated by the heterodimeric transcription factor HIF (hypoxia inducible factor), which is stabilised in hypoxia. In normoxia, the von-Hippel Lindau protein (VHL) targets HIF for degradation. Mutation in the VHL gene, as is frequently seen in clear cell renal cell cancer (CCRCC), results in constitutive over-expression of HIF and its gene targets, leading to a pro-angiogenic and pro-tumourigenic state. This thesis examined the expression of hypoxia-regulated miRNAs in cancer. The principal aims were to determine gene targets of miR-210, and to explore the effects of its over-expression and knock-down, both in vitro and in vivo. The expression of hypoxia-regulated miRNAs was examined in clinical renal tumour samples with matched normal tissue controls, and correlated with VHL mutation status. It was found that miR-210 targeted the iron sulphur cluster homologue (ISCU) gene, and was responsible for much of its down-regulation in hypoxia. Knock-down of ISCU had consequences on cell metabolism, in particular involving mitochondrial function and iron metabolism. miR-210 was found to be highly over-expressed in clear cell renal tumours (CCRCC), with greater expression seen in tumours with VHL mutations. miR-210 over-expression was also observed in papillary renal tumours, but to a lesser extent than in CCRCCs. miR-210 expression appeared to be correlated with reduced stage and grade, and improved survival. ISCU protein expression in CCRCCs was determined by immunohistochemistry, which showed that its expression correlated negatively with miR-210 expression, suggesting a functional role of miR-210 in vivo.

Characterized by the severe progressive wastage of skeletal muscle, Duchenne muscular dystrophy (DMD) is a crippling X-linked recessive disease that is caused by the absence of the protein dystrophin. This thesis aimed to critically evaluate the potential of different therapeutic options to combat this disease. Utrophin is a paralogue of dystrophin. The Fiona mouse is an mdx (dystrophin-deficient) transgenic mouse that overexpresses the full-length utrophin protein in skeletal muscle, and various studies have shown that it does not display a dystrophic phenotype. However, these studies have only been performed on sedentary mice. In this work it is demonstrated that utrophin’s protective effect is partially diminished after a sustained period of exercise-induced stress, highlighting for the first time a functional difference between dystrophin and utrophin. This thesis also presents results of two mdx mouse drug trials testing the ameliorative effects of the administration of the drugs GW501516 and C1100, which show that treatment with both drugs results in partial amelioration of the dystrophic phenotype. GW501516 administration results in a beneficial fast-to-slow fibre type switch and an in vivo increase in utrophin protein levels. We have also shown that C1100 treatment results in a significant increase in utrophin A promoter activity in vitro, and the mechanism of action of this drug on this promoter has been deciphered. The global dysregulation of microRNAs in skeletal muscle of mdx and dko (dystrophin- and utrophin-deficient) mice was evaluated by microarray analysis to identify microRNAs involved in the dystrophic pathological cascades. The results of detailed expression analyses of miR-31, miR-206 and miR-503 are presented, and two therapeutically-relevant predicted targets of miR-503 were validated. Overall, this thesis evaluates the potential of different and possibly complementary therapeutic options to combat DMD.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder caused by absence of functional dystrophin protein. This thesis describes investigations into the role of small non-coding RNAs in both DMD pathology, and as potential therapeutic molecules. MicroRNAs (miRNAs) are a class of small RNAs that regulate gene expression and are implicated in wide-ranging cellular processes and pathological conditions. This study has compared differential miRNA expression in proximal and distal limb muscles, diaphragm, heart and serum in the mdx dystrophic mouse model relative to wild-type controls. Global transcriptome analysis revealed muscle-specific patterns of differential miRNA expression as well as commonalities between tissues, including previously identified dystromirs. miR-1, miR-133a and miR-206 were found to be highly abundant in mdx serum, suggesting that these miRNAs are promising disease biomarkers. Indeed, the relative serum levels of these miRNAs were normalised in response to peptide-PMO mediated dystrophin restoration therapy. This study has revealed further complexity in the miRNA transcriptome of the mdx mouse, an understanding of which will be valuable for the development of novel DMD therapeutics and for monitoring their efficacy. Myostatin is a secreted growth factor that negatively regulates muscle mass and is therefore a potential pharmacological target for the treatment of muscle wasting disorders such as DMD. This study describes a novel myostatin inhibition approach in which small interfering RNAs (siRNAs) complementary to a promoter-associated transcript induce transcriptional gene silencing (TGS) in cultured myotubes. Silencing was sensitive to treatment with the histone deacetylase inhibitor Trichostatin A, and the silent state chromatin mark H3K9me2 was enriched at the myostatin promoter following siRNA transfection, suggesting epigenetic remodelling underlies the silencing effect. These observations suggest that long-term epigenetic silencing may be feasible for myostatin and that TGS is a promising novel therapeutic strategy for the treatment of muscle wasting disorders. The work in this thesis therefore demonstrates the potential of small RNAs as therapeutic agents and as disease biomarkers in the context of DMD.

MicroRNAs represent a special class of small (~21–25 nucleotides) non-coding RNA molecules which exert powerful post-transcriptional control over gene expression in eukaryotes. Indeed microRNAs likely represent the most abundant class of regulators in animal gene regulatory networks. This study describes the recovery and network analyses of a suite of homologous microRNA targets recovered through two different prediction methods for whole gene regions across twelve Drosophila species. Phylogenetic criteria under an accepted tree topology were used as a reference frame to 1) make inference into microRNA-target predictions, 2) study mathematical properties of microRNA-gene regulatory networks, 3) and conduct novel phylogenetic analyses using character data derived from weighted edges of the microRNA-target networks. This study investigates the evidences of natural selection and phylogenetic signatures inherent within the microRNA regulatory networks and quantifies time and mutation necessary to rewire a microRNA regulatory network. Selective factors that appear to operate upon seed aptamers include cooperativity (redundancy) of interactions and transcript length. Topological analyses of microRNA regulatory networks recovered significant enrichment for a motif possessing a redundant link in all twelve species sampled. This would suggest that optimization of the whole interactome topology itself has been historically subject to natural selection where resilience to attack have offered selective advantage. It seems that only a modest number of microRNA–mRNA interactions exhibit conservation over Drosophila cladogenesis. The decrease in conserved microRNA-target interactions with increasing phylogenetic distance exhibited a cure typical of a saturation phenomena. Scale free properties of a network intersection of microRNA target predictions methods were found to transect taxonomic hierarchy.

Hepatitis C virus recurrence (which is defined by detection of HCV RNA in serum) in post-transplanted liver is universal but the progression of infection remains unpredictable, varying from case to case. It has been estimated that 75%-80% of the HCV recurrence patients will suffer chronic hepatitis C infection and up to a third of them will progress into the development of fibrosis and cirrhosis within 5 years post-transplantation. Therefore, finding ways to predict early on the progression of fibrosis can contribute to better prognoses. Recent literatures have mentioned that the hepatitis C virus relies on the host microRNA-122 (miR-122) for assistance in replication of the viral genome in hepatocytes. Experimental depletion of miRNA-122 in the cell line Huh 7 has shown up to an 80% decrease in HCV whereas an increase of miRNA-122 has shown an increase of HCV. Since miRNAs are known to have numerous indirect roles by the binding of the target messenger RNAs (mRNAs) and repressing the expression of their proteins, we hypothesized that the elucidation of associations between miRNA-122 and the histological severity in HCV recurrence post-liver transplantation might serve as a biomarker in predicting the outcome of HCV recurrence severity in patients. We also evaluated the expression levels of BCAP31 (a predicted target of miRNA-122), and CD4 (T cell surface molecules involved in immune response) among the HCV recurrence severity groups. RNA samples were isolated from FFPE liver samples from patients with HCV recurrence post-transplantation, and Reverse Transcription and TaqMan Real-Time PCR were carried out for qualitative analysis. We did not see any association between the levels of miRNA-122 expression and severity of HCV recurrence, but we did find a positive correlation between the miRNA-122 expression and the HCV viral load in Group 3 (Severe) at time of HCV recurrence, which supports previous studies of the role of miRNA-122 in HCV replication. We did not find any associations between the expression of BCAP31 and the severity of HCV recurrence but we did discovery an inverse relationship between miRNA-122 and BCAP31 in Group 3 (Severe) at time of HCV recurrence, confirming our assumption of miRNA:mRNA interaction. Also, we did find CD4 expression being statistically significant between Group 1 (Benign) versus Group 3 (Severe), which may support the hypothesis that strong, adequate CD4+ T-cell response is associated with better outcome post-liver transplantation.

Prostate cancer is the most common cancer occurring in males. The identification of novel microRNAs (miRs) that contribute to tumor progression represents prospective treatment targets. miRs are small non-coding RNAs important in gene regulation with specific tissue expression patterns. Each miR is thought to affect the expression of hundreds of different RNA targets. Two putative oncomiRs, miR-155 and miR-146a, were shown to be differentially expressed in the human derived, prostate cell sublines M12 and F6. Quantification of endogenous miR expression showed high levels in the metastatic M12 cell line versus low in its weakly tumorigenic F6 variant. The restoration of miR expression to M12 levels was evaluated on F6 growth, morphology, and in vitro behavior. F6 plus miR-155 or miR-146a displayed increased growth, motility and invasiveness when compared to M12, with less organized structural morphology when grown embedded in matrigel. Altogether these results suggest that the overexpression of miRs 155 and 146a could contribute to tumor progression in vivo.

<p>Immunity is broadly defined as a mechanism of protection against non-self entities, a process which must be sufficiently robust to both eliminate the initial foreign body and then be maintained over the life of the host. Life-long immunity is impossible without the development of immunological memory, of which a central component is the cellular immune system, or T cells. Cellular immunity hinges upon a naïve T cell pool of sufficient size and breadth to enable Darwinian selection of clones responsive to foreign antigens during an initial encounter. Further, the generation and maintenance of memory T cells is required for rapid clearance responses against repeated insult, and so this small memory pool must be actively maintained by pro-survival cytokine signals over the life of the host.</p><p>T cell development, function, and maintenance are regulated on a number of molecular levels through complex regulatory networks. Recently, small non-coding RNAs, miRNAs, have been observed to have profound impacts on diverse aspects of T cell biology by impeding the translation of RNA transcripts to protein. While many miRNAs have been described that alter T cell development or functional differentiation, little is known regarding the role that miRNAs have in T cell maintenance in the periphery at homeostasis. </p><p>In Chapter 3 of this dissertation, tools to study miRNA biology and function were developed. First, to understand the effect that miRNA overexpression had on T cell responses, a novel overexpression system was developed to enhance the processing efficiency and ultimate expression of a given miRNA by placing it within an alternative miRNA backbone. Next, a conditional knockout mouse system was devised to specifically delete miR-191 in a cell population expressing recombinase. This strategy was expanded to permit the selective deletion of single miRNAs from within a cluster to discern the effects of specific miRNAs that were previously inaccessible in isolation. Last, to enable the identification of potentially therapeutically viable miRNA function and/or expression modulators, a high-throughput flow cytometry-based screening system utilizing miRNA activity reporters was tested and validated. Thus, several novel and useful tools were developed to assist in the studies described in Chapter 4 and in future miRNA studies. </p><p>In Chapter 4 of this dissertation, the role of miR-191 in T cell biology was evaluated. Using tools developed in Chapter 3, miR-191 was observed to be critical for T cell survival following activation-induced cell death, while proliferation was unaffected by alterations in miR-191 expression. Loss of miR-191 led to significant decreases in the numbers of CD4+ and CD8+ T cells in the periphery lymph nodes, but this loss had no impact on the homeostatic activation of either CD4+ or CD8+ cells. These peripheral changes were not caused by gross defects in thymic development, but rather impaired STAT5 phosphorylation downstream of pro-survival cytokine signals. miR-191 does not specifically inhibit STAT5, but rather directly targets the scaffolding protein, IRS1, which in turn alters cytokine-dependent signaling. The defect in peripheral T cell maintenance was exacerbated by the presence of a Bcl-2YFP transgene, which led to even greater peripheral T cell losses in addition to developmental defects. These studies collectively demonstrate that miR-191 controls peripheral T cell maintenance by modulating homeostatic cytokine signaling through the regulation of IRS1 expression and downstream STAT5 phosphorylation.</p><p>The studies described in this dissertation collectively demonstrate that miR-191 has a profound role in the maintenance of T cells at homeostasis in the periphery. Importantly, the manipulation of miR-191 altered immune homeostasis without leading to severe immunodeficiency or autoimmunity. As much data exists on the causative agents disrupting active immune responses and the formation of immunological memory, the basic processes underlying the continued maintenance of a functioning immune system must be fully characterized to facilitate the development of methods for promoting healthy immune function throughout the life of the individual. These findings also have powerful implications for the ability of patients with modest perturbations in T cell homeostasis to effectively fight disease and respond to vaccination and may provide valuable targets for therapeutic intervention.</p> / Dissertation

PhD, Faculty of Health Sciences, University of the Witwatersrand, 2009 / Chronic infection with the hepatitis B virus (HBV) is a major risk factor for cirrhosis and hepatocellular carcinoma, which is the sixth most common cancer worldwide. Available treatment for chronic HBV infection has limited efficacy in preventing associated complications. The compact and multifunctional nature of the viral genome limits its mutability making HBV an ideal candidate for therapy based on nucleic acid hybridisation. The potent and specific gene silencing that can be achieved with RNA interference (RNAi) has fueled interest in exploiting this pathway as a therapeutic modality. Synthetic and expressed RNA sequences have been used to activate RNAi. These engineered sequences mimic natural substrates of the RNAi pathway, which allows them to enter and reprogramme the pathway to effect silencing of intended targets. Tradionally expressed RNAi activators have been transcribed as short hairpin RNA (shRNA) sequences from RNA polymerase III (Pol III) promoters. These shRNA mimic precursor microRNA (pre-miRNA) and consequently enter the RNAi pathway at a relatively late stage. Overexpression of shRNA sequences from Pol III promoters, specifically the U6 promoter, has been associated with toxic side effects and has raised concerns about the use of expressed RNAi activators. Another concern of developing therapeutic RNAi expression cassettes is the emergence of HBV mutants that are resistant to silencing by a single expressed RNAi effecter. These points have highlighted the need for the development expressed RNAi activators that are effective at low concentrations and capable of combinatorial silencing. To address these issues the aim of this study was to assess the feasibility of anti HBV effecter sequences that mimic an early substrate (viz. primary miRNA or pri-miRNA) of the RNAi pathway. Pri-miRNA expression is typically under the transcriptional control of Pol II promoters. Consequently RNAi activators that Abstract - xi - mimic pri-miRNA, so-called pri-miR shuttles, may be expressed from Pol II promoters. Initially a panel of shRNA expression cassettes driven by a Pol III promoter was constructed and silencing of HBV replication assessed. Pri-miR shuttles were then designed by incorporating guide sequences of the most effective anti HBV U6 shRNA into naturally occurring pri-miR-122 and pri-miR-31. Potent inhibition of viral replication was observed with both Pol III and Pol II-driven pri-miR shuttle expression cassettes in vitro and in vivo. Subsequently liver-specific pri-miR-122 and multimeric pri-miR-31 shuttle expression cassettes were created. Pri-miR-122 shuttle sequences expressed from the alpha-1 antitrypsin promoter and HBV basic core promoter exhibited the best liver-specific silencing. Polycistronic pri-miR-31 shuttle sequences were shown to produce multiple RNAi activators capable of silencing multiple target sequences. Silencing by the pri-miR shuttle sequences was independent of toxic effects that arise from induction of the interferon response or saturation of the endogenous miRNA pathway. Pri-miR shuttles clearly represent an improved option for the use of expressed shRNA and brings therapeutic RNAi technology a step closer to clinical application.

Coprinopsis cinerea is extensively used as a model to study the development of homobasidiomycete fungi. Unraveling the molecular basis of fungal developmental processes would contribute to evolutionary studies, improve the knowledge about multicellularity development, and lead to improvement in the breeding and cultivation of edible or medical homobasidiomycete mushrooms. / I studied the fungal developmental processes in two aspects: 1) the hypothesis of a “developmental hourglass”, and 2) the existence of microRNA-like RNA (milRNA) genes in fungi. / The model of “developmental hourglass” suggests that middevelopment is the most conserved and the most resistant to evolutionary changes. Although extensively explored in animals and plants, such hourglass pattern has not been reported in fungi yet. I tested the hourglass model in C. cinerea using two complementary approaches, the transcriptome age index (TAI) and the transcriptome divergence index (TDI). Both the TAI and the TDI profiles displayed an hourglass pattern over the development of C. cinerea; the young fruiting body stage was the waist that expressed the evolutionarily oldest transcriptome (lowest TAI) and gave the strongest signal of purifying selection (lowest TDI). By cross-kingdom comparisons, it is found that all three kingdoms displayed high expression levels of genes in “information storage and processing” at the waist stage, while genes in “metabolism” became more active later; besides, genes at the waist stage were underrepresented in “signal transduction mechanisms”. / MicroRNA (miRNA) is a group of endogenous non-coding regulatory RNAs of ~22 nt that regulate gene expression in various biological processes such as cell differentiation, development regulation and heterochromatin formation. Past research work reported several simple filamentous fungi to contain milRNAs in their genomes; however, no milRNA has been reported in mushrooms so far. Through computational prediction, I identified 16 putative milRNA genes in C. cinerea. Besides, evolutionary analysis showed that C. cinerea contains Dicer-like proteins (DCLs), which may play roles in milRNA biogenesis. / Both the discovery of a “developmental hourglass” and milRNA genes laid a foundation for analysis of fruiting body formation in fungi, and for evolutionary analysis of multicellular development across kingdoms. / 灰蓋鬼傘（Coprinopsis cinerea）作為一個典型的生物模型，被廣泛地運用在擔子菌生長發育的研究中。從分子層面上解析出真菌生長發育的過程，可以促進進化生物學的研究，提高對多細胞生物演化的認知，以及改善食用菌和藥用菌的育種與培養。 / 我從兩個角度去分析了真菌的生長發育過程：1）真菌的“發育沙漏”假說，2）真菌基因組裡類微RNA（microRNA-like RNA [milRNA]）的基因。 / “發育沙漏”模型指出，發育中期在進化中是最保守的、最能抵抗進化帶來的改變。這個沙漏模型在動物與植物中被廣泛地研究與證實，但是迄今為止，並未有人對其在真菌中的存在進行過探究。我以C. cinerea作為研究模型，採用了兩種互補的方法，transcriptome age index (TAI) 和 transcriptome divergence index (TDI)，探究了發育沙漏在真菌中的存在。兩個指數均在C. cinerea發育過程中呈現出了沙漏的形狀；年輕的子實體（young fruiting body）階段是沙漏的腰身，表達出進化中最古老的基因並給出了最強的淨化選擇（purifying selection）的信號。我將三大真核生物進行了比較，發現在腰身階段時，在“信息儲存和處理”中發揮作用的基因呈現出了高表達量，而“代謝”基因的表達量在後期更高；而且在腰身階段表達的基因中，負責“信號傳遞機制”的基因偏少。 / 微RNA（microRNA [miRNA]）是一類長約22個核苷酸、不轉譯蛋白質的RNA分子。它們可以調節其他基因的表達，在多方面的生理過程中發生作用，比如細胞分化、發育調節、異染色質的形成等等。近幾年的研究表明，一些簡單的絲狀真菌的基因組中含有類微RNA（milRNA）的基因；但是，至今未有報導說蘑菇是否含有此類基因。我運用計算預測的方法在C. cinerea的基因組裡找到了16個可能的類微RNA基因。此外，從進化的角度分析，我發現C. cinerea含有類Dicer蛋白酶（Dicer-like proteins [DCLs]），而這些類Dicer蛋白酶可能在milRNA的產生過程中發揮作用。 / 這篇論文所報導的兩個發現──真菌的“發育漏斗“和類微RNA基因，均為之後子實體發育的研究及多細胞發育的研究奠定了基礎。 / Cheng, Xuanjin. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references. / Abstracts also in Chinese. / Title from PDF title page (viewed on 09, September, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.