MicroRNA mir-16 is anti-proliferative in enterocytes and exhibits diurnal rhythmicity in intestinal crypts |
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Authors: | Anita Balakrishnan Adam T. Stearns Peter J. Park Jonathan M. Dreyfuss David B. Rhoads Ali Tavakkolizadeh |
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Affiliation: | a Department of Surgery, Brigham and Women's Hospital, Boston, MA 02115, USAb Department of Surgery, Harvard Medical School, Boston, MA 02115, USAc School of Clinical Sciences, Division of Gastroenterology, University of Liverpool, Liverpool L69 3GE, UKd Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 2JD, UKe Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115, USAf Harvard Medical School, Center for Biomedical Informatics, Boston, MA 02115, USAg Pediatric Endocrine Unit, MassGeneral Hospital for Children, Boston, MA 02114, USA |
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Abstract: | Background and aimsThe intestine exhibits profound diurnal rhythms in function and morphology, in part due to changes in enterocyte proliferation. The regulatory mechanisms behind these rhythms remain largely unknown. We hypothesized that microRNAs are involved in mediating these rhythms, and studied the role of microRNAs specifically in modulating intestinal proliferation.MethodsDiurnal rhythmicity of microRNAs in rat jejunum was analyzed by microarrays and validated by qPCR. Temporal expression of diurnally rhythmic mir-16 was further quantified in intestinal crypts, villi, and smooth muscle using laser capture microdissection and qPCR. Morphological changes in rat jejunum were assessed by histology and proliferation by immunostaining for bromodeoxyuridine. In IEC-6 cells stably overexpressing mir-16, proliferation was assessed by cell counting and MTS assay, cell cycle progression and apoptosis by flow cytometry, and cell cycle gene expression by qPCR and immunoblotting.Resultsmir-16 peaked 6 hours after light onset (HALO 6) with diurnal changes restricted to crypts. Crypt depth and villus height peaked at HALO 13-14 in antiphase to mir-16. Overexpression of mir-16 in IEC-6 cells suppressed specific G1/S regulators (cyclins D1-3, cyclin E1 and cyclin-dependent kinase 6) and produced G1 arrest. Protein expression of these genes exhibited diurnal rhythmicity in rat jejunum, peaking between HALO 11 and 17 in antiphase to mir-16.ConclusionsThis is the first report of circadian rhythmicity of specific microRNAs in rat jejunum. Our data provide a link between anti-proliferative mir-16 and the intestinal proliferation rhythm and point to mir-16 as an important regulator of proliferation in jejunal crypts. This function may be essential to match proliferation and absorptive capacity with nutrient availability. |
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Keywords: | Adcy6, adenylate cyclase type 6 BCL2, B-cell lymphoma 2 BrdU, 5-bromo-2-deoxyuridine Ccnd1, cyclin D1 Ccnd2, cyclin D2 Ccnd3, cyclin D3 Ccne1, cyclin E1 Cdk4, cyclin-dependent kinase 4 Cdk6, cyclin-dependent kinase 6 HALO, hours after light onset 3&prime UTR, 3&prime untranslated region |
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