Genomes and Genes
Developing miRNA diagnostic methods and identifying tumor regulatory networks
Principal Investigator: Thomas Tuschl
Abstract: DESCRIPTION (provided by applicant): This application addresses Broad Challenge Area (08) Genomics and Specific Challenge Topic 08-CA-103: Micro-RNAs in Cancer. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by reducing stability and/or translation of fully or partially sequence-complementary target mRNAs (Eulalio et al., 2008;Friedman et al., 2009). Several hundred miRNA gene families have been identified in mammals (Landgraf et al., 2007), many of which are expressed in specific tissues and have been found to be dysregulated in tumors (reviewed in Ventura and Jacks, 2009). miRNA profiles have been correlated to particular tumors, based on their cell of origin, and in some cases to prognosis (Lu et al., 2005;He et al., 2005;Yanaihara et al., 2006). Because miRNAs alter the expression of multiple genes and thereby may be tuning multiple regulatory steps in disease pathways, they represent interesting drug targets;antisense oligonucleotide targeting experiments in mice (Krutzfeldt et al., 2005) and non-human primates (Elmen et al., 2008) have demonstrated the feasibility of manipulating miRNA levels. At the same time, elucidating miRNA target networks may shed important insights for defining regulatory processes contributing to cancer. The overall aim of this proposal is to advance small RNA profiling and small RNA histological methods for cost-effective characterization of large numbers of patient samples. To interpret the alterations observed in miRNA expression patterns, we will apply our recently developed powerful biochemical approach for direct sequencing of miRNA-targeted mRNA networks to relevant tumor cell culture models. Finally, these recent developments will be implemented in breast cancer research analyzing 177 histologically and clinically characterized tumor samples. First, RNA-deep-sequencing-based methods will be to a stage where they are suitable to define miRNA expression patterns and mutational status for large numbers of clinical samples with limited material. Second, alterations detected in miRNA expression patterns will be followed by miRNA in situ hybridization, for which we recently developed new small RNA fixation techniques and overcame the biggest obstacle hindering miRNA histological detection in tissue sections. Third, to gain insights into the biology of miRNA deregulation, we will apply our recently developed Photoreactive-Uridine-Enhanced Crosslinking and Immunoprecipitation (PURE- CLIP) method of miRNA-bound Argonaute protein complexes to identify the miRNA target sites in transcriptomes of tumor cells. In the last aim, we will apply our methods to assess the value of miRNA analysis in a large collection of triple negative (HER2 gene amplification, Estrogen Receptor (ER), and Progesterone Receptor (PR) negative) and other types of breast tumors in collaboration with Marc van de Vijver at the Academic Medical Center in Amsterdam, The Netherlands. The specimen collection includes 80 triple negative, 51 HER2 positive, 26 ER positive, 11 normal and 20 ductal carcinoma in situ (DCIS) samples. These specimens are already characterized histologically, as well as at the mRNA transcript expression level, and patient clinical characteristics and outcome are available. We will determine absolute miRNA content, relative miRNA composition or profile, miRNA mutational status, and miRNA expression patterns and assess correlations with already determined clinical parameters. We will also define the miRNA-target RNA regulatory networks in tumor cells to be able to interpret consequences of miRNA deregulation in breast cancer. Developing miRNA diagnostic methods and identifying miRNA regulatory networks in tumors PUBLIC HEALTH RELEVANCE: miRNAs are recently identified small non-coding RNAs that regulate many developmental and physiological processes including cancer. This proposal aims to develop new small RNA cloning protocols to meet requirements in clinical research, enhance in situ hybridization methods for cellular localization of miRNAs and at the same time adapt these methods for use in automated platforms of diagnostic laboratories, and clarify miRNA regulatory networks in cancer cell lines, by adapting our recently developed method for identification of miRNA mRNA targets.
Funding Period: ----------------2009 - ---------------2011-
more information: NIH RePORT
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Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065, USA
Adv Exp Med Biol 774:1-20. 2013..Given the critical role miRNAs play in tumorigenesis processes and their disease specific expression, they hold potential as therapeutic targets and novel biomarkers...
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Laboratory of RNA Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA
Methods 58:94-105. 2012....
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Laboratory of RNA Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, Box 186, NY 10065, USA
Methods 58:164-70. 2012..Here we present a step-by-step protocol for generating barcoded small RNA cDNA libraries compatible with Illumina HiSeq sequencing, thereby facilitating miRNA and other small RNA profiling of large sample collections...
- Quantitative mass spectrometry and PAR-CLIP to identify RNA-protein interactionsMarion Scheibe
Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
Nucleic Acids Res 40:9897-902. 2012..We report near perfect agreement between the two approaches. Nevertheless, they are non-redundant, and ideally complement each other to map the RNA-protein interaction network...
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Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, 1230 York Avenue, Box 186, New York, NY 10065, USA
Methods 58:171-87. 2012....
- The viral and cellular microRNA targetome in lymphoblastoid cell linesRebecca L Skalsky
Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina, United States of America
PLoS Pathog 8:e1002484. 2012..This comprehensive survey of the miRNA targetome in EBV-infected B cells represents a key step towards defining the functions of EBV-encoded miRNAs, and potentially, identifying novel therapeutic targets for EBV-associated malignancies...
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Department of Microbiology Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA
Cell Host Microbe 10:515-26. 2011..In summary, this study identifies an extensive list of KSHV miRNA targets, which are likely to influence viral replication and pathogenesis...
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Howard Hughes Medical Institute, Laboratory for RNA Molecular Biology, The Rockefeller University, New York, New York 10065, USA
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Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York 10065, USA
Cancer Res 71:4443-53. 2011....
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Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, NY 10065, USA
Genes Chromosomes Cancer 50:338-47. 2011..These results suggest that the powerful promoters of FET genes are predominantly responsible for the oncogenic effect of transcription factor translocations in sarcomas...
- miRNAs in human cancerThalia A Farazi
Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, Rockefeller University, New York, NY 10065, USA
J Pathol 223:102-15. 2011..Given the critical role miRNAs play in tumorigenesis processes and their disease-specific expression, they hold potential as therapeutic targets and novel biomarkers...