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

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.

SUBMITTER: Bar M

PROVIDER: S-EPMC2847579 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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

Bar Merav M 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

Stem cells (Dayton, Ohio) 20080626 10

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 nov ...[more]

PMID: 18583537

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Project description:BACKGROUND: MicroRNAs are a class of small non-coding RNAs that regulate mRNA expression at the post - transcriptional level and thereby many fundamental biological processes. A number of methods, such as multiplex polymerase chain reaction, microarrays have been developed for profiling levels of known miRNAs. These methods lack the ability to identify novel miRNAs and accurately determine expression at a range of concentrations. Deep or massively parallel sequencing methods are providing suitable platforms for genome wide transcriptome analysis and have the ability to identify novel transcripts. RESULTS: The results of analysis of small RNA sequences obtained by Solexa technology of normal peripheral blood mononuclear cells, tumor cell lines K562 and HL60 are presented. In general K562 cells displayed overall low level of miRNA population and also low levels of DICER. Some of the highly expressed miRNAs in the leukocytes include several members of the let-7 family, miR-21, 103, 185, 191 and 320a. Comparison of the miRNA profiles of normal versus K562 or HL60 cells revealed a specific set of differentially expressed molecules. Correlation of the miRNA with that of mRNA expression profiles, obtained by microarray, revealed a set of target genes showing inverse correlation with miRNA levels. Relative expression levels of individual miRNAs belonging to a cluster were found to be highly variable. Our computational pipeline also predicted a number of novel miRNAs. Some of the predictions were validated by Real-time RT-PCR and or RNase protection assay. Organization of some of the novel miRNAs in human genome suggests that these may also be part of existing clusters or form new clusters. CONCLUSIONS: We conclude that about 904 miRNAs are expressed in human leukocytes. Out of these 370 are novel miRNAs. We have identified miRNAs that are differentially regulated in normal PBMC with respect to cancer cells, K562 and HL60. Our results suggest that post - transcriptional processes may play a significant role in regulating levels of miRNAs in tumor cells. The study also provides a customized automated computation pipeline for miRNA profiling and identification of novel miRNAs; even those that are missed out by other existing pipelines. The Computational Pipeline is available at the website: http://mirna.jnu.ac.in/deep_sequencing/deep_sequencing.html.

Dataset Information

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

Publications

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

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