RESUMO
BACKGROUND: MicroRNAs (miRNAs) are important for the development and function of neutrophils. miR-130a is highly expressed during early neutrophil development and regulates target proteins important for this process. miRNA targets are often identified by validating putative targets found by in silico prediction algorithms one at a time. However, one miRNA can have many different targets, which may vary depending on the context. Here, we investigated the effect of miR-130a on the proteome of a murine and a human myeloid cell line. RESULTS: Using pulsed stable isotope labelling of amino acids in cell culture and mass spectrometry for protein identification and quantitation, we found 44 and 34 proteins that were significantly regulated following inhibition of miR-130a in a miR-130a-overexpressing 32Dcl3 clone and Kasumi-1 cells, respectively. The level of miR-130a inhibition correlated with the impact on protein levels. We used RAIN, a novel database for miRNA-protein and protein-protein interactions, to identify putative miR-130a targets. In the 32Dcl3 clone, putative targets were more up-regulated than the remaining quantified proteins following miR-130a inhibition, and three significantly derepressed proteins (NFYC, ISOC1, and CAT) are putative miR-130a targets with good RAIN scores. We also created a network including inferred, putative neutrophil miR-130a targets and identified the transcription factors Myb and CBF-ß as putative miR-130a targets, which may regulate the primary granule proteins MPO and PRTN3 and other proteins differentially expressed following miR-130a inhibition in the 32Dcl3 clone. CONCLUSION: We have experimentally identified miR-130a-regulated proteins within the neutrophil proteome. Linking these to putative miR-130a targets, we provide an association network of potential direct and indirect miR-130a targets that expands our knowledge on the role of miR-130a in neutrophil development and is a valuable platform for further experimental studies.
Assuntos
MicroRNAs/genética , Neutrófilos/metabolismo , Proteoma , Animais , Linhagem Celular , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Camundongos , Células Mieloides/metabolismo , Proteômica/métodosRESUMO
Smad4 is important in the TGF-ß pathway and required for transcriptional activation and inhibition of cell growth after TGF-ß1 stimulation. We demonstrate that miR-130a is differentially expressed during granulopoiesis and targets Smad4 mRNA. The transcript for Smad4 is present throughout neutrophil maturation, but Smad4 protein is undetectable in the most immature cells, where miR-130a is highly expressed. Two miR-130a binding sites were identified in the 3'-untranslated region of the Smad4 mRNA. Overexpression of miR-130a in HEK293, A549, and 32Dcl3 cells repressed synthesis of Smad4 protein without affecting Smad4 mRNA level. Repression of Smad4 synthesis in a granulocytic cell line by miR-130a reduced its sensitivity to TGF-ß1-induced growth inhibition. This effect was reversed by inhibiting the activity of miR-130a with an antisense probe or by expressing a Smad4 mRNA lacking miR-130a binding sites. High endogenous miR-130a and Smad4 mRNA levels and low expression of Smad4 protein were found in the t(8;21)(q22;q22) acute myelogenous leukemia-derived cell line Kasumi-1. When miR-130a was inhibited by an antisense RNA, the amount of Smad4 protein increased in Kasumi-1 cells and rendered it susceptible for TGF-ß1-mediated cell growth inhibition. Our data indicate that miR-130a is involved in cell cycle regulation of granulocytic cells through engagement of Smad4 in the TGF-ß pathway.
Assuntos
Regulação para Baixo , Células Precursoras de Granulócitos/efeitos dos fármacos , MicroRNAs/genética , Proteína Smad4/genética , Fator de Crescimento Transformador beta1/farmacologia , Regiões 3' não Traduzidas/genética , Doença Aguda , Animais , Sítios de Ligação/genética , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células Precursoras de Granulócitos/metabolismo , Células HEK293 , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , MicroRNAs/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad4/metabolismoRESUMO
BACKGROUND: For most sequenced prokaryotic genomes, about a third of the protein coding genes annotated are "orphan proteins", that is, they lack homology to known proteins. These hypothetical genes are typically short and randomly scattered throughout the genome. This trend is seen for most of the bacterial and archaeal genomes published to date. RESULTS: In contrast we have found that a large fraction of the genes coding for such orphan proteins in the Methanopyrus kandleri AV19 genome occur within two large regions. These genes have no known homologs except from other M. kandleri genes. However, analysis of their lengths, codon usage, and Ribosomal Binding Site (RBS) sequences shows that they are most likely true protein coding genes and not random open reading frames. CONCLUSIONS: Although these regions can be considered as candidates for massive lateral gene transfer, our bioinformatics analysis suggests that this is not the case. We predict many of the organism specific proteins to be transmembrane and belong to protein families that are non-randomly distributed between the regions. Consistent with this, we suggest that the two regions are most likely unrelated, and that they may be integrated plasmids.
