MicroRNA discovery and profiling in human embryonic stem cells by deep sequencing of small RNA libraries.
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.
Bar
Merav
M
Divisions of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
Wyman
Stacia K
SK
Fritz
Brian R
BR
Qi
Junlin
J
Garg
Kavita S
KS
Parkin
Rachael K
RK
Kroh
Evan M
EM
Bendoraite
Ausra
A
Mitchell
Patrick S
PS
Nelson
Angelique M
AM
Ruzzo
Walter L
WL
Ware
Carol
C
Radich
Jerald P
JP
Gentleman
Robert
R
Ruohola-Baker
Hannele
H
Tewari
Muneesh
M
D001483:Base Sequence; D002454:Cell Differentiation; D002460:Cell Line; D030541:Databases, Genetic; D053595:Embryonic Stem Cells / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D015723:Gene Library; D006801:Humans; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D039902:Multipotent Stem Cells / Q000166:cytology / Q000378:metabolism; D009690:Nucleic Acid Conformation; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D043244:Ribonuclease III / Q000378:metabolism; D017423:Sequence Analysis, RNA; D014157:Transcription Factors / Q000378:metabolism
D035683:MicroRNAs; D034741:RNA, Small Interfering; D014157:Transcription Factors; D043244:Ribonuclease III