Dokument: Aberrant expression of microRNA in gliomas: Molecular mechanisms, functional consequences and clinical significance
| Titel: | Aberrant expression of microRNA in gliomas: Molecular mechanisms, functional consequences and clinical significance | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=21736 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20120628-081144-9 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Dr. Liesenberg, Franziska [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Guido Reifenberger [Gutachter] Prof. Dr. Willbold, Dieter [Gutachter] | |||||||
| Stichwörter: | microRNA | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibungen: | Zusammenfassung
MicroRNAs (miRNAs) sind kleine, nicht-kodierende RNAs, welche die Genexpression auf post-transkriptionaler Ebene regulieren. Die Genregulation erfolgt dabei durch die Bindung der miRNA an die 3’-untranslatierte Region (3’-UTR) der mRNA von Zielgenen, welche je nach Komplementarität der Bindesequenz und der beteiligten Proteine entweder an der Translation gehemmt oder vorzeitig abgebaut werden. Aktuelle Studien belegen die kritische Rolle von miRNAs für die Regulierung fundamentaler zellulärer Mechanismen wie Entwicklung, Zellwachstum, Proliferation, Differenzierung von Zell - oder Gewebetypen, Migration von Zellpopulationen und Stoffwechselprozessen. Dysfunktionen von miRNAs können verschiedene Krankheiten zur Folge haben, darunter Tumoren unterschiedlichen Ursprungs einschließlich der glialen Hirntumoren. Hinsichtlich der Tumorigenese können miRNAs entweder eine tumorhemmende (tumorsuppressive) oder tumorfördernde (onkogene) Wirkung zeigen. In der vorliegenden Arbeit wurde die Expression von 365 miRNAs mittels quantitativer PCR-Analysen in vier Glioblastomzelllinien (A172, U138MG, T98G, TP365MG) nach Behandlung der Zellen mit dem DNA-demethylierenden Agens 5-Aza-2'- Deoxycytidin (5-Aza) und dem Histondeacetylase-Inhibitor Trichostatin A (TSA) im Vergleich zu unbehandelten Zellen untersucht. Insgesamt wiesen 50 der untersuchten miRNAs ein signifikant erhöhtes Expressionsniveau in mindestens zwei von vier behandelten Zelllinien im Vergleich zu den zugehörigen nicht behandelten Zelllinien auf. Zwei dieser miRNAs, miR-132 und miR-126, zeigten ein signifikant reduziertes Expressionsniveau in astrozytären Tumoren im Vergleich zu nicht-neoplastischem Hirngewebe. Deshalb wurden diese beiden miRNAs für weitere Analysen ausgewählt und zunächst die verstärkte Expression von miR-132 und miR-126 in unabhängigen Validierungsgruppen 5-Aza/TSA-behandelter Glioblastomzellen bestätigt. Zur Identifizierung epigenetischer Veränderungen, die zu einer verminderten Expression dieser miRNAs in astrozytären Tumoren führen könnten, wurden die 5`genomischen Regionen der beiden miRNAs auf eine aberrante DNA-Methylierung sowie Veränderungen der Histonacetylierung hin untersucht. Mittels Chromatin-Immunopräzipitation (ChIP) und Natriumbisulfit-Sequenzierung konnte nachgewiesen werden, dass sowohl eine veränderte Acetylierung der Histone H3 und H4 sowie eine aberrante DNA Methylierung in der 5`genomischen Region beider untersuchten microRNAs zu einer transkriptionellen Herunterregulation von miR-132 und miR-126 in Glioblastomzellen führte. Funktionelle Analysen der beiden miRNAs in vitro erbrachten einen miRNA-spezifischen Einfluss auf die Apoptoserate von transient mit entsprechenden Vorläufer-miRNAs transfizierten A172 und T98G Gliomzelllen. Für miR-132 wurden JARID1A und SIRT1 als direkt durch miR-132 regulierte Zielgene identifiziert und mittels 3´UTR Luciferase-Reportergen-Assays validiert. Darüber hinaus wurden Hypoxie-bedingte Veränderungen im Expressionsniveau von 365 miRNAs in vier verschiedenen Glioblastom-Stammzelllinien untersucht. Eine signifikant verstärkte Expression unter Hypoxie wurde für acht miRNAs nachgewiesen. Unabhängige Validierungsexperimente bestätigten einen Hypoxie-induzierten Expressionsanstieg von miR-210, welcher durch HIF-1α vermittelt wurde. GPD1L und COX10 wurden als unmittelbare Zielgene von miR-210 in Gliomzellen identifiziert und mit Hilfe von 3`UTR Luciferase-Reportergen-Assays bestätigt. Zusammenfassend unterstützen die eigenen Ergebnisse eine wichtige Rolle von miRNAs in der Pathogenese von Gliomen. Es wurde gezeigt, dass eine aberrante DNA-Methylierung und Histonmodifikationen zu einer verminderten Expression von miR-132 und miR-126 in Gliomen führen. Zusätzlich konnten mit SIRT1 und JARID1A zwei interessante Zielgene von miR-132 in Gliomen charakterisiert werden. Des Weiteren wurde eine über HIF-1a vermittelte Hochregulation von miR-210 in Gliomen gefunden, die wiederum zu einer verminderten Expression der miR-210 Zielgene GPD1L und COX10 führt. Die immer offensichtlicher werdende Rolle von miRNAs in der Entstehung und Progression von Gliomen rechtfertigt weiterführende Untersuchungen, um ein besseres molekulares Verständnis, eine genauere Diagnostik und neue Therapieansätze für diese bislang unheilbaren Tumoren zu finden.Abstract MicroRNAs (miRNAs) are small, non-coding RNAs that are effective post-transcriptional regulators of gene expression. Binding of miRNAs to the 3`-untranslated region (3`-UTR) of messenger RNAs (mRNAs) results in translational inhibition or enhanced mRNA cleavage. MiRNAs play an important role in the regulation of fundamental cellular mechanisms, such as cell proliferation, differentiation, apoptosis, cell migration and metabolism. They are also involved in the pathogenesis of human diseases including cancer. In this study, the expression of 365 distinct human miRNAs was determined by stem-loop real-time reverse transcriptase PCR in four established glioblastoma cell lines (A172, U138MG, T98G, TP365MG) treated with the demethylating agent 5-aza-2`-deoxycytidine (5-Aza) and the histone deacetylase inhibitor trichostatin A (TSA), and compared to the miRNA expression profiles in the respective untreated control cell lines. Thereby, 50 miRNAs were identified that demonstrated an increased expression (fold change ≥ 2) in at least two of the four 5-Aza/TSA-treated glioma cell lines compared to the non-treated control cells. Two of these miRNAs, miR-132 and miR-126, were significantly down-regulated in astrocytic tumors relative to non-neoplastic brain tumors and were therefore selected for independent validation and further molecular characterization using sequencing of sodium bisulfite-modified DNA and chromatin immunoprecipitation analysis of the 5´-genomic regions of each miRNA. These studies revealed that modified acetylation of the core histones H3 and H4 might cause reduced expression of miR-132 and miR-126 in gliomas either alone or in conjunction with 5`CpG island hypermethylation of the miRNAs. Overexpression of miR-132 and miR-126 precursors in glioma cells increased apoptotic activity. Gene expression profiling was carried out to identify miR-132 and miR-126 regulated targets. Using Western-blot analysis and 3’-UTR luciferase assays, SIRT1 and JARID1A were identified as direct targets of miR-132. The second part of this doctoral thesis addressed the identification of miRNAs induced by hypoxia that may have relevance in glioma pathogenesis. A total of eight miRNAs were found to be significantly up-regulated by hypoxia in a study of four glioblastoma stem cell lines grown under hypoxic versus normoxic conditions. Independent validation experiments revealed that hypoxia induced expression of miR-210 in glioblastoma cell lines via HIF-1α. MiR-210 down-regulated the expression of GPD1L and COX10 by directly binding to their 3`UTRs. Taken together, the results summarized in this thesis provide new insights into the involvement of miRNAs in the pathogenesis of human gliomas. The epigenetic alterations leading to transcriptional downregulation of miR-132 and miR-126 in gliomas were clarified and SIRT1 was validated as a direct target of these miRNAs Moreover, hypoxia-mediated regulation of miR-210 via HIF-1α was characterized in glioma cells, and GPD1L and COX10 were identified as immediate targets of miR-210. | |||||||
| Quelle: | References
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References to web pages 3.1.2.2 Real-time PCR analysis using TaqMan® miRNA assays. https://products.appliedbiosystems.com 02.02.2012 Table 17: SYBR® Green Dye assay chemistry. http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/real- time-pcr/taqman-and-sybr-green-chemistries.html 22.03.2012 Figure 5: Conversion of unmethylated cytosine to uracil by sodium bisulfite treatment. http://www.cmmt.ubc.ca/sites/default/files/pdf_methylseqr_protocol.pdf 29.03.2012 Figure 7: Schematic representation of the SDS-PAGE. http://www.imb-jena.de/~rake/Bioinformatics_WEB/proteins_purification.html 01.04.2012 Figure 8: Schematic representation of a western blot transfer. http://technologyinscience.blogspot.de/2011/12/ western-blot-protein-immunoblot.html 03.04.2012 Figure 9: Chemiluminescent detection of Western blot. http://advansta.com/Chemiluminescent_Western_Detection.html 13.04.2012 Figure 10: Cleavage of the non - fluorescent caspase substrate Z-DEVD-R110. http://www.promega.com Apo-ONE® Homogeneous Caspase-3/7 Assay Technical Bulletin 14.04.2012 Figure 11: Reaction of Luciferin and ATP to Oxyluciferin, ADP and light. http://www.promega.com CellTiter-Glo® Luminescent Cell Viability Assay Technical Bulletin 14.04.2012 Figure 14: Schematic representation of the bioluminescent reaction catalyzed by Firefly and Renilla luciferases http://www.promega.com Dual-Glo® Luciferase Assay System Technical Manual 14.04.2012 4.6 Identification and validation of putative miR-132/miR-212, miR-126 and miR-210 targets http://diana.pcbi.upenn.edu/miRGen/v3/miRGen.html (version v3) 01.05.2012 microRNA.org software (version: August 2010) 01.05.2012 4.1.2 Epigenetically regulated miRNAs in gliomas 4.1.3 MiRNAs induced by hypoxia in glioblastoma stem cell lines http://genome.ucsc.edu/ (version: February 2009) 07.04.2012 3.5 3`- Luciferase reporter gene assay system http://www.starseq.com 05.04.2012 | |||||||
| Rechtliche Vermerke: | Ehrenwörtliche Erklärung
Hiermit erkläre ich, dass ich die vorliegende Dissertation eigenständig und ohne unerlaubte Hilfe angefertigt und diese in der vorgelegten oder in ähnlicher Form noch bei keiner anderen Institution eingereicht habe. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Bezug: | Institut für Neuropathologie Düsseldorf
Räumliche Erstreckung: 2008-2012 | |||||||
| Fachbereich / Einrichtung: | Medizinische Fakultät » Institute » Institut für Neuropathologie | |||||||
| Dokument erstellt am: | 28.06.2012 | |||||||
| Dateien geändert am: | 28.06.2012 | |||||||
| Promotionsantrag am: | 09.05.2012 | |||||||
| Datum der Promotion: | 19.06.2012 |
