Journal
STEM CELLS
Volume 26, Issue 10, Pages 2496-2505Publisher
WILEY
DOI: 10.1634/stemcells.2008-0356
Keywords
MicroRNA; Embryonic stem cells; Deep sequencing; Pyrosequencing; 454 sequencing
Categories
Funding
- Oncology Training [5 T32 CA009515-21/22]
- Career Development in Pediatric and Medical Oncology award National Cancer Institute [(NCI)/5 K12 CA076930]
- Chromosome Metabolism Training [5 T32 CA09657-16]
- Interdisciplinary Training in Cancer Research [CA80416]
- National Institute of Environmental Health Sciences [P30 ES07033, P41 HG004059-01]
- National Institute of General Medical Sciences (NIGMS) [P20 GM069983-01]
- NIGMS [P01 GM081619-01]
- Tietze Award
- NIH
- March of Dimes
- NIH/NCI Cancer Center Support [5 P30 CA015704]
- Fred Hutchinson Cancer Research Center
- Roche Diagnostics
- NATIONAL CANCER INSTITUTE [T32CA009657, K12CA076930, T32CA009515, T32CA080416, P30CA015704] Funding Source: NIH RePORTER
- NATIONAL HUMAN GENOME RESEARCH INSTITUTE [P41HG004059] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES [P30ES007033] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [P20GM069983, R01GM083867, P01GM081619] Funding Source: NIH RePORTER
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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. STEM CELLS 2008; 26: 2496-2505
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