RESUMO
MicroRNAs (miRNAs) have been described to simultaneously inhibit hundreds of targets, albeit to a modest extent. It was recently proposed that there could exist more specific, exceptionally strong binding to a subgroup of targets. However, it is unknown, whether this is the case and how such targets can be identified. Using Argonaute2-ribonucleoprotein immunoprecipitation and in vivo competitive binding assays, we demonstrate for miRNAs-21, -199-3p and let-7 exceptional regulation of a subset of targets, which are characterized by preferential miRNA binding. We confirm this finding by analysis of independent quantitative proteome and transcriptome datasets obtained after miRNA silencing. Our data suggest that mammalian miRNA activity is guided by preferential binding of a small set of 3'-untranslated regions, thereby shaping a steep gradient of regulation between potential targets. Our approach can be applied for transcriptome-wide identification of such targets independently of the presence of seed complementary sequences or other predictors.
Assuntos
Regiões 3' não Traduzidas/genética , Proteínas Argonautas/metabolismo , Regulação da Expressão Gênica , Imunoprecipitação/métodos , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Inativação Gênica , Humanos , Camundongos , Células NIH 3T3 , Proteoma , RNA Mensageiro/genética , Especificidade por Substrato , TranscriptomaRESUMO
A precise understanding of the role of miR-223 in human hematopoiesis and in the pathogenesis of acute myeloid leukemia (AML) is still lacking. By measuring miR-223 expression in blasts from 115 AML patients, we found significantly higher miR-223 levels in patients with favorable prognosis, whereas patients with low miR-223 expression levels were associated with worse outcome. Furthermore, miR-223 was hierarchically expressed in AML subpopulations, with lower expression in leukemic stem cell-containing fractions. Genetic depletion of miR-223 decreased the leukemia initiating cell (LIC) frequency in a myelomonocytic AML mouse model, but it was not mandatory for rapid-onset AML. To relate these observations to physiologic myeloid differentiation, we knocked down or ectopically expressed miR-223 in cord-blood CD34⺠cells using lentiviral vectors. Although miR-223 knockdown delayed myeloerythroid precursor differentiation in vitro, it increased myeloid progenitors in vivo following serial xenotransplantation. Ectopic miR-223 expression increased erythropoiesis, T lymphopoiesis, and early B lymphopoiesis in vivo. These findings broaden the role of miR-223 as a regulator of the expansion/differentiation equilibrium in hematopoietic stem and progenitor cells where its impact is dose- and differentiation-stage-dependent. This also explains the complex yet minor role of miR-223 in AML, a heterogeneous disease with variable degree of myeloid differentiation.
Assuntos
Sangue Fetal/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/metabolismo , MicroRNAs/biossíntese , Neoplasias Experimentais/metabolismo , Células-Tronco Neoplásicas/metabolismo , RNA Neoplásico/biossíntese , Adulto , Animais , Proliferação de Células/genética , Eritropoese/genética , Feminino , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Linfopoese/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , MicroRNAs/genética , Pessoa de Meia-Idade , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Células-Tronco Neoplásicas/patologia , RNA Neoplásico/genéticaRESUMO
MicroRNAs (miRNAs) are key physiological regulators in multiple cell types. Here, we assessed platelet production and function in mice deficient in miR-223, one of the most abundantly expressed miRNAs in platelets and megakaryocytes. We found platelet number, size, life-span as well as surface expression of platelet adhesion receptors to be unchanged in miR-223-deficient mice. Likewise, loss of miR-223 did not affect platelet activation, adhesion and aggregation and also had no effect on bleeding times. Moreover, miR-223 null megakaryocytes developed normally and were capable to form pro-platelets. However, we detected a transient delay in the recovery of platelet numbers following antibody-induced platelet depletion in miR-223-deficient animals. This delay was not observed after transplantation of bone marrow from miR-223-deficient animals into wild-type recipients, indicating a non-cell-autonomous role of miR-223 for thrombopoiesis. Overall, our data indicate a surprisingly modest role of miR-223 in platelet production, while the function of platelets does not seem to depend on miR-223.
Assuntos
Plaquetas/fisiologia , Megacariócitos/fisiologia , MicroRNAs/metabolismo , Animais , Citotoxicidade Celular Dependente de Anticorpos/genética , Tempo de Sangramento , Coagulação Sanguínea/genética , Transplante de Medula Óssea , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Contagem de Células , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , MicroRNAs/genética , Ativação Plaquetária/genética , Trombopoese/genética , Quimeras de TransplanteRESUMO
BACKGROUND: Several microRNAs (miRs) have been shown to regulate gene expression in the heart, and dysregulation of their expression has been linked to cardiac disease. miR-378 is strongly expressed in the mammalian heart but so far has been studied predominantly in cancer, in which it regulates cell survival and tumor growth. METHODS AND RESULTS: Here, we report tight control of cardiomyocyte hypertrophy through miR-378. In isolated primary cardiomyocytes, miR-378 was found to be both necessary and sufficient to repress cardiomyocyte hypertrophy. Bioinformatic prediction suggested that factors of the mitogen-activated protein kinase (MAPK) pathway are enriched among miR-378 targets. Using mRNA and protein expression analysis along with luciferase assays, we validated 4 key components of the MAPK pathway as targets of miR-378: MAPK1 itself, insulin-like growth factor receptor 1, growth factor receptor-bound protein 2, and kinase suppressor of ras 1. RNA interference with these targets prevented the prohypertrophic effect of antimiR-378, suggesting their functional relation with miR-378. Because miR-378 significantly decreases in cardiac disease, we sought to compensate for its loss through adeno-associated virus-mediated, cardiomyocyte-targeted expression of miR-378 in an in vivo model of cardiac hypertrophy (pressure overload by thoracic aortic constriction). Restoration of miR-378 levels significantly attenuated thoracic aortic constriction-induced cardiac hypertrophy and improved cardiac function. CONCLUSIONS: Our data identify miR-378 as a regulator of cardiomyocyte hypertrophy, which exerts its activity by suppressing the MAPK signaling pathway on several distinct levels. Restoration of disease-associated loss of miR-378 through cardiomyocyte-targeted adeno-associated virus-miR-378 may prove to be an effective therapeutic strategy in myocardial disease.
Assuntos
Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , MicroRNAs/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Transdução de Sinais/fisiologia , Adenoviridae/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo/fisiologia , Proteína Adaptadora GRB2/antagonistas & inibidores , Proteína Adaptadora GRB2/fisiologia , MicroRNAs/genética , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteínas Quinases/fisiologia , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Receptores de Somatomedina/antagonistas & inibidores , Receptores de Somatomedina/fisiologiaRESUMO
MicroRNAs (miRNAs) are small non-coding RNAs that control expression of complementary target mRNAs. A growing number of miRNAs has been implicated in the pathogenesis of cardiac diseases, mostly based not on functional data, but on the observation that they are dysregulated in diseased myocardium. Consequently, our knowledge regarding a potential cardiac role of the majority of miRNAs is limited. Here, we report the development of an assay format that allows the simultaneous analysis of several hundred molecules with regard to their phenotypic effect on primary rat cardiomyocytes. Using automated microscopy and an edge detection algorithm, this assay achieved high reproducibility and a robust assessment of cardiomyocyte size as a key parameter. Screening a library of synthetic miRNAs revealed several miRNAs previously not recognized as pro- or anti-hypertrophic. Out of these, we selected nine miRNAs and confirmed the pro-hypertrophic potential of miR-22, miR-30c, miR-30d, miR-212, miR-365 and the anti-hypertrophic potential of miR-27a, miR-27b and miR-133a. Quantitative analysis of the expression level of pro-hypertrophic miRNAs in primary cardiomyocytes indicated a rather low level of correlation of the phenotypic effects of individual miRNAs and their expression level. This assay allows the automated determination of cell size in primary cardiomyocytes and permitted the identification of a set of miRNAs capable of regulating cardiomyocyte hypertrophy. Elucidating their mechanism of action should provide insight into mechanisms underlying the cardiomyocyte hypertrophic response. This article is part of a Special Issue entitled 'Possible Editorial'.