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 highquality 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 genomewide 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.

Determination of the Human Cardiomyocyte mRNA and miRNA Differentiation Network by Fine-Scale Profiling

“…3B), as expected [39,40]. Although the levels of the miRNAs never returned to baseline, even at 120 days, such residual miR-302 expression has been observed in other differentiation experiments [39,41,42]. The let-7 family of miRNAs, which reinforce the commitment to differentiation [43], also exhibited the expected expression pattern with the levels increasing during differentiation (Supplementary Fig.…”

Identification of Resting and Type I IFN-Activated Human NK Cell miRNomes Reveals MicroRNA-378 and MicroRNA-30e as Negative Regulators of NK Cell Cytotoxicity

“…In-depth miRNA profiling by deep sequencing with high-resolution views of expressed miRNAs over a wide dynamic range of expression levels has already been realized in several kinds of tissues and cell types, including human embryonic stem cells (25), human B cells (26), cell types during mouse lymphopoiesis (17), human renal cell carcinoma (27), and mouse leukemia (28). The study that revealed dynamic miRNomes during lymphopoiesis selected mouse adherent lymphokine-activated NK cells (ALAK, NK cells) as a mouse NK cell substitute to get the mouse NK cell miRNome (17), which may affect the biological and physiological significance of this mouse NK cell miRNome.…”

Characterization of microRNAs Involved in Embryonic Stem Cell States

“…Previously, miRNA expression has been examined in murine and human ES cells using cloning, microarray, quantitative polymerase chain reaction (PCR), and deep sequencing technologies on selected cell lines [20][21][22][23][24][25][26][27][28][29]. These studies typically involve embryoid body (EB) formation as an intermediate step, and the differentiation protocols extend for several weeks.…”