RESUMO
New biomarkers of drug-induced liver injury (DILI) are required in the clinic and in preclinical pharmaceutical evaluation. Liver-enriched microRNAs are promising serum biomarkers of acetaminophen-induced acute liver injury in mice. The utility of circulating microRNAs as biomarkers of human acute DILI is discussed in the context of correlation with existing biomarkers of liver injury and patient outcomes in acetaminophen toxicity, mechanisms of cellular microRNA release, and their potential advantages over current clinical biomarkers of DILI.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/sangue , MicroRNAs/sangue , Animais , Biomarcadores/sangue , Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Fígado/efeitos dos fármacos , CamundongosRESUMO
Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance process that promotes selective degradation of imperfect messages containing premature translation termination codons (PTCs). In yeast, PTCs trigger both deadenylylation-independent mRNA decapping, thereby allowing their rapid degradation by a 5' to 3' exonuclease, and to a smaller extent accelerated deadenylylation. It is not clear to what extent this decay pathway is conserved in higher eukaryotes. We used a transcriptional pulse strategy relying on a tetracycline-regulated promoter to study the decay of a PTC- containing beta-globin mRNA in human cells. We show that a PTC destabilizes the mRNA and decreases its half-life from >16 h to 3 h. The deadenylylation rate is increased, but not sufficiently to account for the decreased half-life on its own. Using a circularization RT-PCR (cRT-PCR) strategy, we could detect decapped degradation intermediates and measure simultaneously their poly(A) tail length. This allowed us to show that a PTC enhances the rate of mRNA decapping and that decapped products have been deadenylylated to a certain extent. Thus the major feature of the NMD pathway, enhanced decapping, is conserved from yeast to man even though the kinetic details might differ between various mRNAs and/or species.
Assuntos
Códon sem Sentido/genética , Globinas/genética , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , Estabilidade de RNA/genética , Engenharia Genética , Meia-Vida , Células HeLa , Humanos , Cinética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Oestrogen deficiency enhances bone osteoclastogenesis and bone resorption. Evidence of cooperation between stromal cells and osteoclast precursors in mice suggests that oestradiol acts by regulating cytokine release from stromal cells. Bone marrow stroma contains multipotent progenitors that give rise to many mesenchymal lineages, including osteoblasts that may regulate osteoclast differentiation. We immortalized and characterized six human bone marrow stromal cell lines (presence of Stro1, secretion of alkaline phosphatase, osteocalcin, formation of lipid droplets, and presence of alpha and beta oestrogen receptors). The response of cytokines to oestradiol was then evaluated in vitro, as were the phorbol myristate acetate (PMA)-stimulated cytokine levels. Cells had the characteristics of undifferentiated stromal cells (Stro1+, RANK-L+), and expressed alpha-oestrogen receptors. The osteoblast phenotype (amounts of alkaline phosphatase and osteocalcin) was weak and there was a poor capacity to differentiate into adipocytes. These cell lines did not respond to oestradiol by producing interleukin 6 (IL-6), IL-1 or tumour necrosis factor alpha (TNF-alpha) either constitutively or after stimulation with PMA. Moreover, RANK-L and osteoprotegerin expressions were not regulated by oestradiol in vitro. Thus, modulation of these cytokines by stromal cells do not appear to be the mechanism by which oestradiol regulates bone resorption in humans.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Citocinas/biossíntese , Estradiol/farmacologia , Animais , Proteínas de Transporte/genética , Linhagem Celular , Receptor alfa de Estrogênio , Expressão Gênica/efeitos dos fármacos , Glicoproteínas/genética , Humanos , Interleucina-1/biossíntese , Interleucina-6/biossíntese , Glicoproteínas de Membrana/genética , Camundongos , Osteoprotegerina , Ligante RANK , RNA/genética , RNA/metabolismo , Receptor Ativador de Fator Nuclear kappa-B , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Estrogênio/genética , Receptores do Fator de Necrose Tumoral , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
In yeast, the major mRNA degradation pathway is initiated by poly(A) tail shortening that triggers mRNA decapping. The mRNA is then degraded by 5'-to-3' exonucleolysis. In mammalian cells, even though poly(A) tail shortening also precedes mRNA degradation, the degradation pathway has not been elucidated. We have used a reverse transcription-PCR approach that relies on mRNA circularization to measure the poly(A) tail length of four mammalian mRNAs. This approach allows for the simultaneous analysis of the 5' and 3' ends of the same mRNA molecule. For all four mRNAs analyzed, this strategy permitted us to demonstrate the existence of small amounts of decapped mRNA species which have a shorter poly(A) tail than their capped counterparts. Kinetic analysis of one of these mRNAs indicates that the decapped species with a short poly(A) tail are mRNA degradation products. Therefore, our results indicate that decapping is preceded by a shortening of the poly(A) tail in mammalian cells, as it is in yeast, suggesting that this mRNA degradation pathway is conserved throughout eukaryotic evolution.