RESUMO
A microRNA, termed miR-In300 or miR-3906, suppresses the transcription of myf5 through silencing dickkopf-related protein 3 (dkk3r/dkk3a) during early development when myf5 is highly transcribed, but not at late stages when myf5 transcription is reduced. Moreover, after 24 hpf, when muscle cells are starting to differentiate, Dkk3a could not be detected in muscle tissue at 20 hpf. To explain these reversals, we collected embryos at 32 hpf, performed assays, and identified homer-1b, which regulates calcium release from sarcoplasmic reticulum, as the target gene of miR-3906. We further found that either miR-3906 knockdown or homer-1b overexpression increased expressions of fmhc4 and atp2a1 of calcium-dependent fast muscle fibrils, but not slow muscle fibrils, and caused a severe disruption of sarcomeric actin and Z-disc structure. Additionally, compared to control embryos, the intracellular calcium concentration ([Ca(2+)]i) of these treated embryos was increased as high as 83.9-97.3% in fast muscle. In contrast, either miR-3906 overexpression or homer-1b knockdown caused decreases of [Ca(2+)]i and, correspondingly, defective phenotypes in fast muscle. These defects could be rescued by inducing homer-1b expression at later stage. These results indicate that miR-3906 controls [Ca(2+)]i homeostasis in fast muscle through fine tuning homer-1b expression during differentiation to maintain normal muscle development.
Assuntos
MicroRNAs/genética , Fibras Musculares de Contração Rápida/metabolismo , Músculo Esquelético/metabolismo , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Regiões 3' não Traduzidas/genética , Animais , Animais Geneticamente Modificados , Sítios de Ligação/genética , Cálcio/metabolismo , Diferenciação Celular/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Hibridização In Situ , MicroRNAs/metabolismo , Microscopia Eletrônica de Transmissão , Fibras Musculares de Contração Rápida/citologia , Músculo Esquelético/citologia , Músculo Esquelético/embriologia , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/ultraestrutura , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismoRESUMO
Despite significant advances in the treatment of osteosarcoma (OS), overall survival rate of OS patients has remained relatively constant for over two decades and novel approaches are needed to further improve prognosis. Here, we report the anti-tumor effect of SC-1, a novel sorafenib derivative that closely resembles sorafenib structurally but is devoid of kinase inhibitory activity, on OS cells through mediation of signal transducer and activator of transcription 3 (STAT3). SC-1 showed similar effects to sorafenib on growth inhibition and apoptosis, and downregulated phospho-STAT3 (p-STAT3) at tyrosine 705 in all tested OS cell lines (U2OS, HOS, and 143B). Expression of STAT3-driven genes, including cylcin D1 and c-myc, were also repressed by SC-1. Ectopic expression of STAT3 in 143B cells abolished apoptosis in SC-1-treated cells. Inhibition of SHP-1 decreased SC-1-induced apoptosis. SC-1 upregulated the activity of SHP-1 in tested OS cell lines in a dose-dependent manner. Finally, SC-1 reduced 143B tumor growth significantly in vivo, which was associated with downregulation of p-STAT3 and upregulation of SHP-1 activity. These data demonstrate that SC-1 has clinical potential for the treatment of OS patients.