Article

Ago2 Immunoprecipitation Identifies Predicted MicroRNAs in Human Embryonic Stem Cells and Neural Precursors

Rutgers Stem Cell Research Center, Rutgers University, Piscataway, New Jersey, United States of America.
PLoS ONE (Impact Factor: 3.23). 09/2009; 4(9):e7192. DOI: 10.1371/journal.pone.0007192
Source: PubMed

ABSTRACT

MicroRNAs are required for maintenance of pluripotency as well as differentiation, but since more microRNAs have been computationally predicted in genome than have been found, there are likely to be undiscovered microRNAs expressed early in stem cell differentiation.
SOLiD ultra-deep sequencing identified >10(7) unique small RNAs from human embryonic stem cells (hESC) and neural-restricted precursors that were fit to a model of microRNA biogenesis to computationally predict 818 new microRNA genes. These predicted genomic loci are associated with chromatin patterns of modified histones that are predictive of regulated gene expression. 146 of the predicted microRNAs were enriched in Ago2-containing complexes along with 609 known microRNAs, demonstrating association with a functional RISC complex. This Ago2 IP-selected subset was consistently expressed in four independent hESC lines and exhibited complex patterns of regulation over development similar to previously-known microRNAs, including pluripotency-specific expression in both hESC and iPS cells. More than 30% of the Ago2 IP-enriched predicted microRNAs are new members of existing families since they share seed sequences with known microRNAs.
Extending the classic definition of microRNAs, this large number of new microRNA genes, the majority of which are less conserved than their canonical counterparts, likely represent evolutionarily recent regulators of early differentiation. The enrichment in Ago2 containing complexes, the presence of chromatin marks indicative of regulated gene expression, and differential expression over development all support the identification of 146 new microRNAs active during early hESC differentiation.

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    • "High-throughput sequencing is not only able to reveal the expression profiles of known miRNAs, but also is able to discover new miRNAs that have not been recorded previously in any databases, in particular the miRBase repository. Small RNA sequencing has been used to carry out research on various types of stem cells, including embryonic stem cells [24–27], hematopoietic stem cells [28], and neural precursor cells [24]. Novel miRNAs have also been identified using smRNA-seq during neural differentiation of embryonic stem cells [27] and during endothelial differentiation [29]. "
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    ABSTRACT: Background Endothelial progenitor cells (EPCs) play a fundamental role in not only blood vessel development but also post-natal vascular repair. Currently EPCs are defined as early and late EPCs based on their biological properties and their time of appearance during in vitro culture. Both EPC types assist angiogenesis and have been linked to ischemia-related disorders, including coronary artery disease (CAD). Results We found late EPCs are more mobile than early EPCs and matured endothelial cells (ECs). To pinpoint the mechanism, microRNA profiles of early EPCs late EPCs, and ECs were deciphered by small RNA sequencing. Obtained signatures made up of both novel and known microRNAs, in which anti-angiogenic microRNAs such as miR-221 and miR-222 are more abundant in matured ECs than in late EPCs. Overexpression of miR-221 and miR-222 resulted in the reduction of genes involved in hypoxia response, metabolism, TGF-beta signalling, and cell motion. Not only hamper late EPC activities in vitro, both microRNAs (especially miR-222) also hindered in vivo vasculogenesis in a zebrafish model. Reporter assays showed that miR-222, but not miR-221, targets the angiogenic factor ETS1. In contrast, PIK3R1 is the target of miR-221, but not miR-222 in late EPCs. Clinically, both miR-221-PIK3R1 and miR-222-ETS1 pairs are deregulated in late EPCs of CAD patients. Conclusions Our results illustrate EPCs and ECs exploit unique miRNA modalities to regulate angiogenic features, and explain why late EPC levels and activities are reduced in CAD patients. These data will further help to develop new plasma biomarkers and therapeutic approaches for ischemia-related diseases or tumor angiogenesis. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-802) contains supplementary material, which is available to authorized users.
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    • "MiRNAs regulate multiple cellular processes including embryonic stem cell (ESC) self-renewal [11] and neural differentiation [12]–[17]. Previous work identifying novel miRNAs and their expression profiles has established a distinct subset of miRNAs with enriched or specific expression in neural tissues and neural precursors [18]–[20]. Brain-enriched miRNAs such as miR-9, miR-124a, miR-125, and numerous others are induced in primary neural tissues and differentiating primary neurons [20]–[22]. Conversely, several ESC specific miRNAs are down-regulated during retinoic acid-induced differentiation of neuronal precursor cells [12] consistent with the hypothesis that miRNAs are likely to be key regulators of neural differentiation. "
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    ABSTRACT: MiR-9, a neuron-specific miRNA, is an important regulator of neurogenesis. In this study we identify how miR-9 is regulated during early differentiation from a neural stem-like cell. We utilized two immortalized rat precursor clones, one committed to neurogenesis (L2.2) and another capable of producing both neurons and non-neuronal cells (L2.3), to reproducibly study early neurogenesis. Exogenous miR-9 is capable of increasing neurogenesis from L2.3 cells. Only one of three genomic loci capable of encoding miR-9 was regulated during neurogenesis and the promoter region of this locus contains sufficient functional elements to drive expression of a luciferase reporter in a developmentally regulated pattern. Furthermore, among a large number of potential regulatory sites encoded in this sequence, Mef2 stood out because of its known pro-neuronal role. Of four Mef2 paralogs, we found only Mef2C mRNA was regulated during neurogenesis. Removal of predicted Mef2 binding sites or knockdown of Mef2C expression reduced miR-9-2 promoter activity. Finally, the mRNA encoding the Mef2C binding partner HDAC4 was shown to be targeted by miR-9. Since HDAC4 protein could be co-immunoprecipitated with Mef2C protein or with genomic Mef2 binding sequences, we conclude that miR-9 regulation is mediated, at least in part, by Mef2C binding but that expressed miR-9 has the capacity to reduce inhibitory HDAC4, stabilizing its own expression in a positive feedback mechanism.
    Full-text · Article · Apr 2014 · PLoS ONE
    • "However, mouse and human ES cells do have a different expression profile of miRs. The numbers of homologous miR genes is different and the miR sequences are variable between mice and humans[28,30]. This contributes to the different signaling pathways involved maintenance of pluripotency rather than to evolutionary differences. "

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