RESUMEN
Although microRNAs (miRNAs) are involved in many biological processes, the mechanisms whereby miRNAs regulate osteoblastic differentiation are poorly understood. Here, we found that BMP-4-induced osteoblastic differentiation of bone marrow-derived ST2 stromal cells was promoted and repressed after transfection of sense and antisense miR-210, respectively. A reporter assay demonstrated that the activin A receptor type 1B (AcvR1b) gene was a target for miR-210. Furthermore, inhibition of transforming growth factor-beta (TGF-beta)/activin signaling in ST2 cells with SB431542 promoted osteoblastic differentiation. We conclude that miR-210 acts as a positive regulator of osteoblastic differentiation by inhibiting the TGF-beta/activin signaling pathway through inhibition of AcvR1b.
Asunto(s)
Receptores de Activinas Tipo I/antagonistas & inhibidores , Diferenciación Celular , MicroARNs/metabolismo , Osteoblastos/citología , Receptores de Activinas Tipo I/metabolismo , Animales , Proteína Morfogenética Ósea 4/metabolismo , Células Cultivadas , Ratones , Osteoblastos/metabolismo , Transducción de Señal , Transfección , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Although various microRNAs regulate cell differentiation and proliferation, no miRNA has been reported so far to play an important role in the regulation of osteoblast differentiation. Here we describe the role of miR-125b in osteoblastic differentiation in mouse mesenchymal stem cells, ST2, by regulating cell proliferation. The expression of miR-125b was time-dependently increased in ST2 cells, and the increase in miR-125b expression was attenuated in osteoblastic-differentiated ST2 cells induced by BMP-4. The transfection of exogenous miR-125b inhibited proliferation of ST2 cells and caused inhibition of osteoblastic differentiation. In contrast, when the endogenous miR-125b was blocked by transfection of its antisense RNA molecule, alkaline phosphatase activity after BMP-4 treatment was elevated. These results strongly suggest that miR-125b is involved in osteoblastic differentiation through the regulation of cell proliferation.
Asunto(s)
Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , MicroARNs/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Osteogénesis/fisiología , Animales , Diferenciación Celular , Línea Celular , Proliferación Celular , Regulación hacia Abajo , RatonesRESUMEN
We have developed a microarray-based assay for the genotyping of Staphylococcus aureus strains. A DNA microarray consisting of 221 genes with 390 oligonucleotide probes was designed to identify characteristic genes or gene alleles of S. aureus. The 221 genes were chosen on the basis of the following criteria: (i) genes used as control for the microarray system, (ii) virulence genes, (iii) resistance genes and their regulators, and (iv) genes constituting genomic islands, e.g., SCCmec. The microarray system was established by determining the method to prepare targets by random-primer labeling with chromosomal DNA and the conditions for hybridization. We verified the system by using DNAs of seven strains, the genome of which has been fully sequenced. Furthermore, the presence of 32 genes and the types of SCCmec elements and coagulase genes carried by another 27 strains were examined and compared with the results of PCR. As a result, the presence or absence of 182 genes out of the 221 genes was verified. Our data showed the usefulness of the oligonucleotide microarray based assay in identifying important marker sets, such as toxin genes, resistance genes, SCCmec elements, and coagulase genes, for the molecular epidemiology of S. aureus.
Asunto(s)
Resistencia a la Meticilina , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Staphylococcus aureus/aislamiento & purificación , Técnicas de Tipificación Bacteriana , Coagulasa , Sondas de ADN/metabolismo , Genes Bacterianos , Resistencia a la Meticilina/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/normas , Fenotipo , Reacción en Cadena de la Polimerasa , Secuencias Reguladoras de Ácidos Nucleicos/genética , Pruebas Serológicas , Staphylococcus aureus/clasificaciónRESUMEN
We have developed a unique photo-cross-linking approach for immobilizing a variety of small molecules in a functional-group-independent manner. Our approach depends on the reactivity of the carbene species generated from trifluoromethylaryldiazirine upon UV irradiation. It was demonstrated in model experiments that the photogenerated carbenes were able to react with every small molecule tested, and they produced multiple conjugates in most cases. It was also found in on-array immobilization experiments that various small molecules were immobilized, and the immobilized small molecules retained their ability to interact with their binding proteins. With this approach, photo-cross-linked microarrays of about 2000 natural products and drugs were constructed. This photo-cross-linked microarray format was found to be useful not merely for ligand screening but also to study the structure-activity relationship, that is, the relationship between the structural motif (or pharmacophore) found in small molecules and its binding affinity toward a protein, by taking advantage of the nonselective nature of the photo-cross-linking process.
