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Functional Characterization of Mirnas in Tumour Biology

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Functional Characterization of Mirnas in Tumour Biology Functional characterization of miRNAs in tumour biology Keerthana Krishnan M. Biotech A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2014 Institute for Molecular Bioscience Abstract MicroRNAs are non-coding, negative regulators of gene expression which act either by repressing protein translation or via mRNA degradation. They have been shown to play biologically significant roles in various processes like cell differentiation, proliferation, apoptosis and development in humans as well as other model organisms. miRNAs direct the wide repertoire of normal biological processes by down-regulating the expression of their target genes. Deregulation of miRNAs has been shown to be associated with various human diseases such as cancer. Although several oncomirs have been identified to date, functional studies to understand their mode of action have been hampered by the lack of tools to accurately predict and validate the gene networks repressed. This thesis aims to address this issue by using a combination of high- throughput technologies and subsequent experimental validation using cell based assays. Chapter two takes a closer look at miR-182, which at the time this study was initiated was shown to be deregulated in several cancers, but its biological role and target networks in the context of breast cancer was relatively unknown. We used biotinylated synthetic miRNA to pull-down its endogenous mRNA targets to reveal that it disrupts key pathways underlying tumorigenesis, and subsequently confirmed its clinical relevance in human breast cancers. Chapter three takes a similar approach to identify the biologically relevant targets of miR-139, a novel breast cancer oncomir. The role of miR- 139 is more akin to being a potential tumour suppressor supporting our data and published datasets where its expression is frequently downregulated in human breast cancers. In Chapter four, we take a more global approach where using next-generation sequencing technology we identify miRNAs which show dynamic expression across various phases of the cell cycle, another biological process typically disrupted during tumorigenesis. Using online datasets, we try to identify if there is a significant correlation between these oscillating miRNAs and cancer, and also possible regulators of their expression. These approaches facilitate the identification of novel oncomirs and subsequent characterization of their biologically relevant targets using context-dependent cell i models. In addition these studies show that these miRNAs achieve their functional output by targeting multiple genes, which belong to the same pathway, adding to the existing notion of concomitant suppression. Together, this leads to a better understanding of the miRNA-mediated disruption to specific molecular processes underlying tumorigenesis. Such studies are imperative to explore the potential of oncomirs as possible prognostic, diagnostic or therapeutic tools. ii Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. iii Publications during candidature Peer-reviewed papers Thiagarajan RD, Cloonan N, Gardiner BB, Mercer TR, Kolle G, Nourbakhsh E, Wani S, Tang D, Krishnan K, Georgas KM, Rumballe BA, Chiu HS, Steen JA, Mattick JS, Little MH, Grimmond SM. Refining transcriptional programs in kidney development by integration of deep RNA-sequencing and array-based spatial profiling. BMC Genomics, 2011. 12: p. 441. Cloonan N, Wani S, Xu Q, Gu J, Lea K, Heater S, Barbacioru C, Steptoe AL, Martin HC, Nourbakhsh E, Krishnan K, Gardiner BB, Wang X, Nones K, Steen JA, Matigan N, Wood DLA, Kassahn KS, Waddell N, Shepherd J, Lee C, Ichikawa J, McKernan K, Bramlett K, Kuersten S and Grimmond SM. MicroRNAs and their isomiRs function cooperatively to target common biological pathways. Genome Biol, 2011. 12(12): p. R126. Krishnan K, Steptoe AL, Martin HC, Wani S, Nones K, Waddell N, Mariasegaram M, Simpson PT, Lakhani SR, Gabrielli B, Vlassov A, Cloonan N, Grimmond SM. MicroRNA-182-5p targets a network of genes involved in DNA repair. RNA, 2013. 19(2): p. 230-42 Krishnan K, Steptoe AL, Martin HC, Pattabiraman DR, Nones K, Waddell N, Mariasegaram M, Simpson PT, Lakhani SR, Vlassov A, Grimmond SM, Cloonan N. miR-139-5p is a regulator of metastatic pathways in breast cancer. RNA, 2013. 19(12): p. 1767-80 Pattabiraman DR, McGirr C, Shakhbazov K, Barbier V, Krishnan K, Mukhopadhyay P, Hawthorne P, Trezise AEO, Grimmond SM, Papathanasiou P, Alexander WS, Perkins AC, Levesque JP, Winkler IG, Gonda TJ. Interaction of c-Myb with p300 is required for the induction of acute myeloid leukemia (AML) by human AML oncogenes. Blood, 2014. 123 (17): p. 2682-2690 Martin HC, Wani S, Steptoe AL, Krishnan K, Nones K, Nourbakhsh E, Vlassov A, Grimmond SM and Cloonan N. Imperfect centered miRNA binding sites are common and can mediate functional repression of target mRNAs. Genome Biol, 2014. 15(3): p. R51 Book Chapters Krishnan K, Wood DLA, Steen JA, Grimmond SM and Cloonan N. “Tag Sequencing”. Epigenetic Regulation and Epigenomics – Advances in Molecular Biology and Medicine. 2012 Wiley-Blackwell. p. 145-166 Publications included in this thesis Krishnan K, Steptoe AL, Martin HC, Wani S, Nones K, Waddell N, Mariasegaram M, Simpson PT, Lakhani SR, Gabrielli B, Vlassov A, Cloonan N, Grimmond SM. MicroRNA-182-5p targets a network of genes involved in DNA repair. RNA, 2013. 19(2): p. 230-42: Incorporated as Chapter two Contributor Statement of contribution Author Krishnan K (Candidate) Designed experiments (70%) Performed experiments (70%) Data Analysis (70%) Wrote the paper (60%) Author Steptoe AL Performed experiments (10%) (Stable cell line generation with candidate) Author Martin HC Data Analysis (15%) (Biotin pulldown microarray) Author Wani S Performed experiments (10%) (Biotin pulldown) Author Nones K Performed experiments (7%) (Microarrays) Author Waddell N Designed experiments (5%) Author Mariasegaram M Performed experiments (3%) (Provided RNA from patient samples) Author Simpson PT Wrote the paper (2%) Author Lakhani SR Wrote the paper (2%) Author Gabrielli B Designed experiments (5%) Wrote the paper (5%) Author Vlassov A Provided reagents Author Cloonan N Designed experiments (15%) Data Analysis (15%) Wrote paper (25%) Author Grimmond SM Designed experiments (5%) Wrote paper (6%) vi Krishnan K, Steptoe AL, Martin HC, Pattabiraman DR, Nones K, Waddell N, Mariasegaram M, Simpson PT, Lakhani SR, Vlassov A, Grimmond SM, Cloonan N. miR-139-5p is a regulator of metastatic pathways in breast cancer. RNA, 2013. 19(12): p. 1767-80: Incorporated as Chapter three Contributor Statement of contribution Author Krishnan K (Candidate) Designed experiments (85%) Performed experiments (70%) Data Analysis (75%) Wrote the paper (80%) Author Steptoe AL Performed experiments (10%) (Stable cell line generation with candidate) Author Martin HC Data Analysis (15%) (Biotin pulldown microarray) Author Pattabiraman DR Performed experiments (10%) (Western Blots) Author Nones K Performed experiments (7%) (Microarrays) Author Waddell N Designed experiments (2%) Author Mariasegaram M Performed experiments (3%)(Provided RNA from patient samples) Author Simpson PT Wrote the paper (2%) Author Lakhani SR Wrote the paper (5%) Author Vlassov A Provided reagents Author Grimmond SM Designed experiments (5%) Wrote paper (5%) Author Cloonan N Designed experiments (8%) Data Analysis (10%) Wrote paper (8%) Krishnan K, Wood DLA, Steen JA, Grimmond SM and Cloonan N. “Tag Sequencing”. Epigenetic Regulation and Epigenomics – Advances in Molecular Biology and Medicine. 2012 Wiley-Blackwell. p. 145-166: Incorporated as a section within Chapter one Contributor Statement of contribution Author Krishnan K (Candidate) Wrote the paper (15%) Author Wood DLA Wrote the paper (15%) Author Steen JA Wrote the paper (5%) Author Grimmond SM Wrote and edited paper (5%) Author Cloonan N Wrote and edited paper (60%) vii Contributions by others to the thesis All the work presented in this thesis was performed at the Queensland Centre for Medical Genomics (QCMG) and would not have been possible without the resources provided
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