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* Institute for Research in Biomedicine, Bellinzona, Switzerland;
Immune Disease Institute and Harvard Medical School, Boston, MA 02115; and
Biozentrum, University of Basel, Basel, Switzerland
MicroRNAs (miRNAs) constitute a large family of small noncoding RNAs that have emerged as key posttranscriptional regulators in a wide variety of organisms. Because any one miRNA can potentially regulate expression of a distinct set of genes, differential miRNA expression can shape the repertoire of proteins that are actually expressed during development and differentiation or disease. Here, we have used mast cells as a model to investigate the role of miRNAs in differentiated innate immune cells and found that miR-221–222 are significantly up-regulated upon mast cell activation. Using both bioinformatics and experimental approaches, we identified some signaling pathways, transcription factors, and potential cis-regulatory regions that control miR-221–222 transcription. Overexpression of miR-221–222 in a model mast cell line perturbed cell morphology and cell cycle regulation without altering viability. While in stimulated cells miR-221–222 partially counteracted expression of the cell-cycle inhibitor p27kip1, we found that in the mouse alternative splicing results in two p27kip1 mRNA isoforms that differ in their 3' untranslated region, only one of which is subject to miR-221–222 regulation. Additionally, transgenic expression of miR-221–222 from bacterial artificial chromosome clones in embryonic stem cells dramatically reduced cell proliferation and severely impaired their accumulation. Our study provides further insights on miR-221–222 transcriptional regulation as well as evidences that miR-221–222 regulate cell cycle checkpoints in mast cells in response to acute activation stimuli.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by a start-up grant from the Institute for Research in Biomedicine Foundation to S.M. and National Institutes of Health Grants AI44432 and AI070788 to A.R. M.E.P. is supported by a fellowship from the National Cancer Institute F32 CA126247-01. R.J.M. was temporarily supported by a fellowship from the Ceresio Foundation and is now a recipient of a San Raffaele "Vita e Salute" University predoctoral fellowship.
2 R.J.M. and M.E.P. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Silvia Monticelli, Institute for Research in Biomedicine, Via Vincenzo Vela 6, CH-6500, Bellinzona, Switzerland. E-mail address: silvia.monticelli{at}irb.unisi.ch
4 Abbreviations used in this paper: miRNA, microRNA; BMMC, bone marrow-derived mast cell; CHX, cycloheximide; CsA, cyclosporin A; ES, embryonic stem; HS, hypersensitivity; nt, nucleotide; pri-, primary; qRT-PCR, quantitative RT-PCR; 7AAD, 7-aminoactinomycin D; SCF, stem cell factor; SSC, side scatter; UTR, untranslated region; BAC, bacterial artificial chromosome.
5 The online version of the article contains Extended Methods.
6 The online version of this article contains supplemental material.
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