Plant and animals both contain non-coding small RNAs that play important roles in their growth, development and responses to biotic and abiotic stresses. MicroRNAs are short (20-24nt), endogenously expressed, and a well characterized class of small RNAs, which are derived from processing longer, hairpin-like precursors. Discovery of most miRNAs relies upon either of two methods: i) molecular cloning of small RNAs or ii) prediction of miRNA genes based on conserved sequences, and secondary structures of known miRNAs using computational tools. miRCheck is a popular computational tool chain comprised of various Perl scripts for identifying and profiling plant miRNA genes. The program serves two purposes: 1. Identifying miRNA homologs in target genomic or cDNA sequences using a given small RNA library, 2. Searching for all potential miRNA precursor loci across the target genome based on evolutionarily-conserved structural features without any reference small RNA library to compare with. miRCheck builds upon several popular tools like patscan, RNAfold, einverted, and connects them to provide a complete tool chain for identifying miRNAs. Although miRCheck is a very well designed tool chain, it still has a few issues that need to be addressed to enhance its functionality and efficiency. This work analyzes the working mechanism of miRCheck, proposes some methods to enhance its efficiency and functionality, and implements those in a modified tool chain, py-miRCheck, in Python. To process a long genome sequence, miRCheck looks at small segments and serially evaluates them leading to long run times. Even in a high performance computing node, it takes days to process a standard sized reference genome obtained from NCBI repository. It highlights the inefficiency in the program. On the functionality side, there are several issues that need to be addressed for usability improvements: i) lack of parameterized design, ii) procedural design, iii) lack of GUI interface for running the tool chain, and iv) deployment-related problems. In this work, I address all these areas and also parallelize the tool chain to improve its efficiency by over a factor of 3. I also provide a Django-based prototype web front end to submit queries on a genome sequence. In summary, this work improves the usability of this tool chain to a great extent. / text

Recently, genome-wide surveys for non-coding RNAs have provided evidence for tens of thousands of previously undescribed evolutionary conserved RNAs with distinctive secondary structures. The annotation of these putative ncRNAs, however, remains a difficult problem. Here we describe an SVM-based approach that, in conjunction with a non-stringent filter for consensus secondary structures, is capable of efficiently recognizing microRNA precursors in multiple sequence alignments. The software was applied to recent genome-wide RNAz surveys of mammals, urochordates, and nematodes.

MicroRNAs (miRNAs) are a group of small, ∼21 nt long, riboreg-ulators inhibiting gene expression at a post-transcriptional level. Their most distinctive structural feature is the foldback hairpin of their precursor pre-miRNAs. Even though each pre-miRNA deposited in miRBase has its secondary structure already predicted, little is known about the patterns of structural conservation among pre-miRNAs. We address this issue by clustering the human pre-miRNA sequences based on pairwise, sequence and secondary structure alignment using FOLDALIGN, followed by global multiple alignment of obtained clusters by WAR. As a result, the common secondary structure was successfully determined for four FOLDALIGN clusters: the RF00027 structural family of the Rfam database and three clusters with previously undescribed consensus structures.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2016. / Primary microRNA (pri-miRNA) mimics have been shown to mediate effective gene silencing and are well-suited for therapeutic applications. Pri-miRNA mimics, like natural pri-miRNA, are processed in the endogenous microRNA (miRNA) biogenesis pathway. Elements of the secondary RNA structure are crucial for processing by the Drosha-DGCR8 microprocessor, including a basal stem of - 11 bp. However, structural variation is common and the exact determinants of pri-miRNA processing have been elusive. The aim of this project were to explore the use of natural pri-miRNAs with exceptional basal stem in the design of correspondingmimics and to identify optimal stem features.[Abbreviated Abstract. Open document to view full version] / LG2017

Die Lebertransplantation bietet gegenwärtig die beste Therapiemöglichkeit des Hepatozellulären Karzinoms und der Leberzirrhose im Endstadium. Für die positiven Resultate ist die rechtzeitige Diagnose- und Indikationsstellung entscheidend. Bei einem Japanese Integrated System (JIS) Score von 0 liegt die 5-Jahres-Überlebensrate nach einer Transplantation bei 73 %, bei einem Anstieg des Scores auf den Wert 3, sinkt sie auf 13 %[74]. Die Sensitivität der gegenwärtigen Diagnostik aus bildgebenden Verfahren und der Bestimmung des AFP zur Detektion eines HCC schwankt zwischen 20 % und 94 %[47,48,69]. Der für die Listung zur Transplantation entscheidende MELD-Score vernachlässigt ebenso wie die Milan-Kriterien die genetische und ätiologische Komponente dieser Tumorerkrankung, welche aber maßgeblich das rezidivfreie Überleben bestimmen[16–18,70]. Im Rahmen dieser Dissertation wurden Gewebeproben aus explantierten Lebern auf mikroRNA-Expression untersucht. Des Weiteren wurde zum ersten Mal analysiert, ob mit Hilfe binär-logistischen Regression und der Entscheidungsbaumklassifikation Algorithmen aus mikroRNA-Profilen und laborchemisch-klinischen Parametern zur Detektion und ätiologischen Differenzierung des HCC und der Leberzirrhosen entwickelt werden können. In der vorliegenden Arbeit konnte dargestellt werden, dass zwischen dem HCC-Gewebe und dem tumorumgebenden Gewebe, zwischen tumorumgebendem Gewebe und der reinen Zirrhose sowie zwischen ethyltoxischer und viraler Genese der o.g. Krankheitsbilder die mikroRNAs unterschiedlich stark exprimiert werden. Außerdem konnten aussichtsreiche Modelle berechnet werden, die eine Differenzierung zwischen Tumor und tumorumgebendem Gewebe mit 87,5 %, eine Unterscheidung von tumorumgebender Zirrhose von einer reinen Zirrhose mit 94,3 % bzw. 96,3 % und eine Differenzierung zwischen Tumorgewebe und reiner Zirrhose mit 91,9 % bzw. 92,1% ermöglichen. Die weitere Analyse zeigte, dass die Modelle ebenfalls dazu geeignet sind, die Lebererkrankungen nach der Ätiologie zu differenzieren. Die dargestellten Methoden sind in der Beachtung der mit großen Potenzial[24,30] versehenen mikroRNAs und der laborchemisch-klinischen Parameter neuartige Verfahren, die sowohl für die weitere Grundlagenforschung als auch für die Ergänzung der derzeit etablierten diagnostischen und allokativen Verfahren wichtige Erkenntnisse liefern.

Plattenepithelkarzinome des Kopf-Hals-Bereichs bilden die weltweit sechst-häufigste Gruppe maligner Erkrankungen. Trotz moderner interdisziplinärer und multimodaler Therapie sind die durchschnittlichen 5-Jahres-Überlebensraten mit ca. 50-60 Prozent seit vielen Jahren unverändert niedrig. Es besteht ein großer Bedarf an verlässlichen Biomarkern zur Abschätzung des individuellen Risikos von aggressiven Krankheitsverläufen sowie zur Prognosebestimmung und Therapie-Überwachung. miRNAs sind kleine nicht protein-codierende RNA-Moleküle, deren Funktion in der posttransskriptionalen Genregulation besteht. Diese RNAs können möglicherweise als Biomarker verwendet werden. In dieser Studie sollte daher die Extraktion von 30 microRNAs an 43 Proben formalin-fixierter, in Paraffin eingebetteter (FFPE) Proben von Mundhöhlenkarzinomen vorgenommen werden. Hierzu erfolgte eine Trennung von Tumor und gesundem Gewebe. Außerdem erfolgte eine Korrelationsanalyse der Expressionsdaten mit relevanten klinischen und pathologischen Daten wie Alter, Geschlecht, Tumor-Stadium und Größe. Das Extraktionsverfahren war erfolgreich und es konnten diverse unterschiedlichen Expressionsmuster zwischen Tumor und Vergleichsgewebe festgestellt werden. Außerdem zeigten sich signifikante Korrelationen zwischen den Expressionsdaten und den klinischen Parametern. / Squamous cell carcinoma of the head and neck (HNSCC) is the sixth most common malignancy worldwide. The past decades have not led to substantial improvement in diagnosis and therapy. Analysis of miRNA-expression may help to determine the progression profiles and outcomes of many different diseases, including HNSCC. Therefore, in this investigation, 43 formalin-fixed, paraffin-embedded (FFPE) samples of oral squamous cell carcinoma were micro-dissected, analysed for expression of 30 miRNAs and were compared with non-tumorous tissue. Furthermore, correlation analysis was performed, investigating possible correlations of miRNA-expression and patient or tumour-linked data, such as age, sex, tumour stage and size. miRNA extraction from FFPE samples functioned well for OSCC, and several miRNAs were differently expressed in tumours compared with non-tumorous tissue , indicating their possible utility as biomarkers. Moreover, some miRNAs showed significant correlations with clinical and pathological data.

Regulatory T cells (Tregs) are a subpopulation of T cells with immuno-suppressive properties. Tregs play a key role in immune response regulation and tolerance to antigens, thereby preventing autoimmunity, and may be partly responsible for the lack of an appropriate immune response against tumor cells. However, a human microRNA (miR) Treg signature has not been described yet. We investigated human natural Tregs and identified a signature composed of five microRNAs (-21, -31, -125a, -181c and -374). Among those, two were considerably under-expressed (miR-31 and miR-125a). We identified a functional target sequence for miR-31 in the 3’ untranslated region (3’ UTR) of FOXP3 mRNA. Using lentiviral transduction of fresh cord blood T cells, we demonstrated that miR-31 and miR-21 had opposite effects on FOXP3 expression. We showed that miR-31 negatively regulates FOXP3 expression by binding directly to its potential target site in the 3’ UTR of FOXP3 mRNA. We next demonstrated that miR-21 acted as a positive, though indirect, regulator of FOXP3 expression. Recent reports have shown that histone deacetylase inhibitors increased FOXP3 expression in T cells. We therefore decided to investigate in non-Treg CD4-positive cells, the mechanisms by which an aspecific opening of the chromatin could lead to an increased FOXP3 expression. We focused on the binding of potentially activating transcription factors to the promoter region of FOXP3 and on modifications in the five miRs constituting the Treg signature. Valproate treatment induced binding of Ets-1 and Ets-2 transcription factors to the FOXP3 promoter and acted positively on its expression, by increasing the acetylation of histone H4 lysines. Valproate treatment also induced the acquisition of the miRs of Treg signature. To elucidate whether the changes in the miRs expression could be due to the increased FOXP3 expression, we transduced these non-Tregs with a FOXP3 lentiviral expression vector, and found no changes in miRs expression. Therefore, the modification in their miR expression profile is not due to an increased expression of FOXP3 but directly results from histone deacetylase inhibition. Rather, the increased FOXP3 expression results from the additive effects of Ets factors binding and the change in the expression level of miR-21 and miR-31. These data, by allowing a better understanding of the molecular phenomena underlying the number and function of Tregs, could open the door to novel therapeutic approaches in cancer immunotherapy and treatment of autoimmune disorders.

MicroRNA is a single-stranded RNA molecule of about 22 nucleotides in length and is expressed endogenously. It functions as a gene regulator by pairing imperfectly with 3’ untranslated region (3’UTR) of target mRNAs, leading to translational inhibition. MicroRNA is implicated in many regulatory pathways and hence affects various cellular activities. In the development of cancer, genetic alterations occurred at miRNA locus and its expression level is dysregulated in various cancers versus normal tissue counterparts. It is thus important to find the targets of dysregulated microRNAs contributing to progression of cancer. To facilitate long term functional studies, a microRNA expression construct with unique futures was generated. Stable expression of miR-378 enhanced cell survival, reduced caspase-3 activity, and promoted tumor growth and angiogenesis. By algorithmic predictions and proteomic analysis, two tumor suppressors, SuFu and Fus-1, were found to be translationally regulated by miR-378. Target validation was confirmed by co-transfection experiments and luciferase activity assays, reassuring its oncogenic role by regulating two tumor suppressor genes simultaneously. Conversely, microRNA can also function as a tumor suppressor by modulating expression of Versican, an extracellular matrix protein known to facilitate tumorigenesis and angiogenesis. By a novel PCR method, more than one microRNA were found to bind to Versican 3’UTR. iii Among these microRNAs, targeting of Versican and Fibronectin by miR199a-3p was validated. Expression of a fragment of Versican 3’UTR was expected to antagonize the function of miR-199a-3p. Stable expression of Versican 3’UTR resulted change in cell morphology and increased cell-cell adhesion. Analysis of primary tissues from transgenic mice expressing versican 3’UTR showed an increase expression of Versican and Fibronectin, and organ adhesion was found between liver and its surrounding tissues. In addition, 3’UTR also modulated the level of miR-199a-3p and miR-136, alleviating translation of negative cell cycle regulators, PTEN and Rb1. This resulted in reduced cell proliferation and hence diminished tumor growth. These findings suggest a role of microRNA in tumor growth, providing a valuable target for therapeutic intervention.

Prostate cancer, one of the most common cancers among men, can be relatively harmless or extremely aggressive. The most widely used biomarker for the disease, the PSA test, is not independently diagnostic or prognostic of prostate cancer. One of the main challenges of prostate cancer research is to find reliable and effective prognostic biomarkers that will predict cancer recurrence following surgery, in order to identify clinically significant prostate cancer and improve management of the disease. In recent years, microRNAs (miRNAs) have been identified as master regulators of cellular processes, and dysregulated miRNAs have been associated with cancer development and progression. The intent of my PhD research program was to uncover novel miRNAs that contribute to prostate cancer pathogenesis in order to assess their potential as predictors of clinical progression. By analyzing a large cohort of primary prostate cancer samples, we have discovered that microRNA-221 (miR-221) is associated with metastasis and biochemical recurrence in prostate cancer, and is downregulated in TMPRSS2:ERG fusion gene- positive tumors. In addition, we have determined that microRNA-182 (miR-182) is overexpressed in prostate cancer and is associated with increased metastasis and clinical progression by targeting a tumors suppressor Forkhead box O1 (FOXO1). Overall, this work introduces novel candidate miRNA genes and downstream targets that are aberrantly expressed in more aggressive prostate cancer, and presents a potentially significant role for miRNAs as prognostic biomarkers that are associated with clinical progression, and perhaps aids in defining how miRNAs might one day serve as anti-cancer therapeutic agents.

MicroRNA is a single-stranded RNA molecule of about 22 nucleotides in length and is expressed endogenously. It functions as a gene regulator by pairing imperfectly with 3’ untranslated region (3’UTR) of target mRNAs, leading to translational inhibition. MicroRNA is implicated in many regulatory pathways and hence affects various cellular activities. In the development of cancer, genetic alterations occurred at miRNA locus and its expression level is dysregulated in various cancers versus normal tissue counterparts. It is thus important to find the targets of dysregulated microRNAs contributing to progression of cancer. To facilitate long term functional studies, a microRNA expression construct with unique futures was generated. Stable expression of miR-378 enhanced cell survival, reduced caspase-3 activity, and promoted tumor growth and angiogenesis. By algorithmic predictions and proteomic analysis, two tumor suppressors, SuFu and Fus-1, were found to be translationally regulated by miR-378. Target validation was confirmed by co-transfection experiments and luciferase activity assays, reassuring its oncogenic role by regulating two tumor suppressor genes simultaneously. Conversely, microRNA can also function as a tumor suppressor by modulating expression of Versican, an extracellular matrix protein known to facilitate tumorigenesis and angiogenesis. By a novel PCR method, more than one microRNA were found to bind to Versican 3’UTR. iii Among these microRNAs, targeting of Versican and Fibronectin by miR199a-3p was validated. Expression of a fragment of Versican 3’UTR was expected to antagonize the function of miR-199a-3p. Stable expression of Versican 3’UTR resulted change in cell morphology and increased cell-cell adhesion. Analysis of primary tissues from transgenic mice expressing versican 3’UTR showed an increase expression of Versican and Fibronectin, and organ adhesion was found between liver and its surrounding tissues. In addition, 3’UTR also modulated the level of miR-199a-3p and miR-136, alleviating translation of negative cell cycle regulators, PTEN and Rb1. This resulted in reduced cell proliferation and hence diminished tumor growth. These findings suggest a role of microRNA in tumor growth, providing a valuable target for therapeutic intervention.