MicroRNA Discovery and Profiling in Human Embryonic Stem Cells by Deep Sequencing of Small RNA Libraries

Stem Cells - Tập 26 Số 10 - Trang 2496-2505 - 2008
Merav Bar1, Stacia K. Wyman2, Brian R. Fritz2, Junlin Qi3,4, Kavita S. Garg5,2, Rachael K. Parkin2, Evan M. Kroh2, Ausra Bendoraite2, Patrick S. Mitchell2, Angelique M. Nelson6,4, Walter L. Ruzzo5,7, Carol B. Ware6,4, Jerald P. Radich1, Robert Gentleman8, Hannele Ruohola‐Baker3,4, Muneesh Tewari1,2
1Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
2Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
3Department of Biochemistry, University of Washington, Seattle, Washington, USA.
4University of Washington Institute for Stem Cell and Regenerative Medicine, Seattle, Washington, USA
5Department of Computer Science and Engineering, University of Washington, Seattle, Washington, USA
6Department of Comparative Medicine, University of Washington, Seattle, Washington, USA
7Department of Genome Sciences, University of Washington, Seattle, Washington, USA
8Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

Tóm tắt

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) was noted to be expressed in undifferentiated hESC and then strongly downregulated with differentiation. Functional annotation analysis of predicted targets of these miRNAs and comparison with 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 with those of recent deep sequencing studies in mouse and human ESC shows 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 they provide a resource for additional studies of miRNA regulation of hESC physiology. Disclosure of potential conflicts of interest is found at the end of this article.

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Stem Cells

Tập 26 Số 10

2496-2505

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