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MicroRNA discovery and profiling in human embryonic stem cells by deep sequencing of small RNA libraries

Bar, Merav and Wyman, Stacia K. and Fritz, Brian R. and Qi, Junlin and Garg, Kavita S. and Parkin, Rachael K. and Kroh, Evan M. and Bendoraite, Ausra and Mitchell, Patrick S. and Nelson, Angelique and Ruzzo, Walter L. and Ware, Carol and Radich, Jerald P. and Gentleman, Robert and Ruohola-Baker, Hannele and Tewari, Muneesh (2008) MicroRNA discovery and profiling in human embryonic stem cells by deep sequencing of small RNA libraries. Stem Cells. ISSN 1066-5099

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Article URL: http://stemcells.alphamedpress.org/cgi/content/abs...

Abstract

We used massively parallel pyrosequencing to discover and characterize microRNAs (miRNAs) expressed in human embryonic stem cells (hESC). Sequencing of small RNA cDNA libraries derived from undifferentiated hESC and from isogenic differentiating cultures yielded a total of 425,505 high-quality sequence reads. A custom data analysis pipeline delineated expression profiles for 191 previously annotated miRNAs, 13 novel miRNAs and 56 candidate miRNAs. Further characterization of a subset of the novel miRNAs in Dicer-knockdown hESC demonstrated Dicer-dependent expression, providing additional validation of our results. A set of 14 miRNAs (9 known and 5 novel) were noted to be expressed in undifferentiated hESC and then strongly down-regulated with differentiation. Functional annotation analysis of predicted targets of these miRNAs and comparison to a null model using non-hESC-expressed miRNAs identified statistically enriched functional categories, including chromatin remodeling and lineage-specific differentiation annotations. Finally, integration of our data with genome-wide chromatin immunoprecipitation data on OCT4, SOX2 and NANOG binding sites implicates these transcription factors in the regulation of nine of the novel/candidate miRNAs identified here. Comparison of our results to those of recent deep sequencing studies in mouse ESC and human ESC show that most of the novel/candidate miRNAs found here were not identified in the other studies. The data indicate that hESC express a larger complement of miRNAs than previously appreciated, and provide a resource for further studies of miRNA regulation of hESC physiology.

Item Type: Article
DOI: 10.1634/stemcells.2008-0356
PubMed ID: 18583537
NIHMSID: NIHMS66861
PMCID: PMC2847579
Grant Numbers: 5 T32 CA009515-21/22 , 5 K12 CA076930 , 5 T32 CA09657-16, CA80416, P30ES07033, P41 HG004059-01 , P20 GM069983-01, P01 GM081619-01 , 5 P30 CA015704
Keywords or MeSH Headings: Base Sequence Cell Differentiation Cell Line Databases, Genetic Embryonic Stem Cells/cytology Embryonic Stem Cells/enzymology Embryonic Stem Cells/metabolism* Expressed Sequence Tags Gene Expression Profiling* Gene Expression Regulation, Developmental Gene Library* Humans MicroRNAs/chemistry MicroRNAs/genetics* Molecular Sequence Data Multipotent Stem Cells/cytology Multipotent Stem Cells/metabolism Nucleic Acid Conformation Pluripotent Stem Cells/cytology Pluripotent Stem Cells/metabolism RNA, Small Interfering/chemistry RNA, Small Interfering/genetics Reproducibility of Results Reverse Transcriptase Polymerase Chain Reaction Ribonuclease III/metabolism Sequence Analysis, RNA* Transcription Factors/metabolism
Subjects: Molecules > RNA > miRNA
Research Methodologies > Genomics > Gene mapping
Cell Types > Stem Cells
Depositing User: Library Staff
Date Deposited: 25 Sep 2008 21:40
Last Modified: 14 Feb 2012 14:42
URI: http://authors.fhcrc.org/id/eprint/26

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