RESUMO
Osteosarcoma (OS) is the most common malignant tumor that develops in bone. Its mortality is very high. Therefore, study of mechanisms of pathogenesis of the OS is urgently required. Previous studies of microarray showed that the expression levels of matrix metallopeptidase 9 (MMP-9) altered significantly in OS. In addition, overexpression of MMP-9 is recognized as an indicator in cancer. However, the exact roles of MMP-9 in OS are not fully investigated. Thus, we firstly studied the roles of MMP-9 in OS and revealed that silence of MMP-9 inhibited OS cell proliferation as determined by MTT assay and colony formation assay. Secondly, we conducted TUNEL assay and confirmed loss of functions of MMP-9 induced OS cell apoptosis. Next, we used lentivector packaging method to overexpress MMP-9 and found that overexpression of MMP-9 promoted OS cell migration. Fourthly, the results of luciferase assay showed that MMP-9 was targeted by hsa-miR-494, which inhibited OS. Fifthly, we revealed that the levels of hsa-miR-494 were upregulated by the drug silybin which inhibited OS. Finally, we revealed that silybin inhibited OS cell viability by altering the protein levels of ß-catenin and Runt-related transcription factor 2 (RUNX2) as determined by western blot and immunocytochemistry (ICC).
Assuntos
Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Metaloproteinase 9 da Matriz/genética , MicroRNAs/genética , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Silimarina/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Silibina , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
MicroRNAs (miRs) play a pivotal role in a variety of biological processes including stem cell differentiation and function. Human foetal femur derived skeletal stem cells (SSCs) display enhanced proliferation and multipotential capacity indicating excellent potential as candidates for tissue engineering applications. This study has examined the expression and role of miRs in human foetal femur derived SSC differentiation along chondrogenic and osteogenic lineages. Cells isolated from the epiphyseal region of the foetal femur expressed higher levels of genes associated with chondrogenesis while cells from the foetal femur diaphyseal region expressed higher levels of genes associated with osteogenic differentiation. In addition to the difference in osteogenic and chondrogenic gene expression, epiphyseal and diaphyseal cells displayed distinct miRs expression profiles. miR-146a was found to be expressed by human foetal femur diaphyseal cells at a significantly enhanced level compared to epiphyseal populations and was predicted to target various components of the TGF-ß pathway. Examination of miR-146a function in foetal femur cells confirmed regulation of protein translation of SMAD2 and SMAD3, important TGF-ß and activin ligands signal transducers following transient overexpression in epiphyseal cells. The down-regulation of SMAD2 and SMAD3 following overexpression of miR-146a resulted in an up-regulation of the osteogenesis related gene RUNX2 and down-regulation of the chondrogenesis related gene SOX9. The current findings indicate miR-146a plays an important role in skeletogenesis through attenuation of SMAD2 and SMAD3 function and provide further insight into the role of miRs in human skeletal stem cell differentiation modulation with implications therein for bone reparation.
Assuntos
Diferenciação Celular/genética , Regulação para Baixo/genética , Feto/citologia , MicroRNAs/metabolismo , Músculo Esquelético/citologia , Proteína Smad2/genética , Proteína Smad3/genética , Células-Tronco/citologia , Regiões 3' não Traduzidas/genética , Sequência de Bases , Sítios de Ligação , Separação Celular , Forma Celular/genética , Condrócitos/metabolismo , Condrócitos/patologia , Condrogênese/genética , Diáfises/citologia , Epífises/citologia , Retroalimentação Fisiológica , Fêmur/embriologia , Perfilação da Expressão Gênica , Humanos , Hipertrofia , MicroRNAs/genética , Dados de Sequência Molecular , Músculo Esquelético/embriologia , Osteogênese/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Células-Tronco/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
The two major aggrecanases involved in osteoarthritis (OA) are ADAMTS-4 and ADAMTS-5. Knock-out studies suggested that ADAMTS-5, but not ADAMTS-4, is the major aggrecanase in murine OA. However, studies of human articular cartilage suggest that ADAMTS-4 also contributes to aggrecan degradation in human OA. This study investigated ADAMTS-4 in human OA. While ADAMTS-4 was virtually absent in control cartilage, numerous ADAMTS-4 immuno-positive chondrocytes were present in OA cartilage and their numbers increased with disease severity. RT-PCR confirmed expression, especially in the surface zone. DNA methylation was lost at specific CpG sites in the ADAMTS-4 promoter in OA chondrocytes, suggesting that the increased gene expression was more than a simple up-regulation, but involved loss of DNA methylation at specific CpG sites, resulting in a heritable and permanent expression of ADAMTS-4 in OA chondrocytes. These results suggest that ADAMTS-4 is epigenetically regulated and plays a role in aggrecan degradation in human OA.
Assuntos
Proteínas ADAM/metabolismo , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Osteoartrite/genética , Osteoartrite/metabolismo , Pró-Colágeno N-Endopeptidase/metabolismo , Proteínas ADAM/genética , Proteína ADAMTS4 , Idoso , Idoso de 80 Anos ou mais , Condrócitos/patologia , Metilação de DNA , Epigênese Genética , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Regulação Enzimológica da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Pró-Colágeno N-Endopeptidase/genéticaRESUMO
OBJECTIVE: To investigate whether the abnormal expression of matrix metalloproteinases (MMPs) 3, 9, and 13 and ADAMTS-4 by human osteoarthritic (OA) chondrocytes is associated with epigenetic "unsilencing." METHODS: Cartilage was obtained from the femoral heads of 16 patients with OA and 10 control patients with femoral neck fracture. Chondrocytes with abnormal enzyme expression were immunolocalized. DNA was extracted, and the methylation status of the promoter regions of MMPs 3, 9, and 13 and ADAMTS-4 was analyzed with methylation-sensitive restriction enzymes, followed by polymerase chain reaction amplification. RESULTS: Very few chondrocytes from control cartilage expressed the degrading enzymes, whereas all clonal chondrocytes from late-stage OA cartilage were immunopositive. The overall percentage of non-methylated sites was increased in OA patients (48.6%) compared with controls (20.1%): 20% versus 4% for MMP-13, 81% versus 47% for MMP-9, 57% versus 30% for MMP-3, and 48% versus 0% for ADAMTS-4. Not all CpG sites were equally susceptible to loss of methylation. Some sites were uniformly methylated, whereas in others, methylation was generally absent. For each enzyme, there was 1 specific CpG site where the demethylation in OA patients was significantly higher than that in controls: at -110 for MMP-13, -36 for MMP-9, -635 for MMP-3, and -753 for ADAMTS-4. CONCLUSION: This study provides the first evidence that altered synthesis of cartilage-degrading enzymes by late-stage OA chondrocytes may have resulted from epigenetic changes in the methylation status of CpG sites in the promoter regions of these enzymes. These changes, which are clonally transmitted to daughter cells, may contribute to the development of OA.
