RESUMO
The aim of the present study was to confirm the role of 11ß-hydroxysteroid dehydrogenases type 2(11ß-HSD-2) in steroid induced osteonecrosis of the femoral head(SANFH). We cultured mouse bone-like cells (MLO-Y4) and mouse osteoblast-like cells (MC3T3-E1). After overexpressed 11ß-HSD-2 successfully, we induced cell apoptosis by dexamethasone (DXM). The level of cell apoptosis, the expression of Bcl-2 in MLO-Y4 cells and the expression of Fas and caspase8 in MC3T3-E1 cells were detected. Then, we constructed 11ß-HSD-2 siRNA plasmid and represented it on MLO-Y4/MC3T3-E1 Cells, to down-regulate the 11ß-HSD-2 expression. After that, we used dexamethasone to induce cell apoptosis. The level of cell apoptosis, the expression of Bcl-2 in MLO-Y4 cells and the expression of Fas and caspase8 in MC3T3-E1 cells were detected again. In the overexpression model of cells, we found that the amount of cell apoptosis, the expression of Fas and caspase8 in MC3T3-E1 cells are lower than that of control groups. The amount of cell apoptosis, the expression of Fas and caspase8 in MC3T3-E1 cells were more than before when we reduced the expression of 11ß-HSD-2. In our study, we concluded that 11ß-HSD-2 plays an important role in the development of bone or osteoblast cell apoptosis, and the decreased expression of 11ß-HSD-2 may aggravate steroid induced bone/osteoblast cell apoptosis.
Assuntos
11-beta-Hidroxiesteroide Desidrogenases/genética , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Osteoblastos/efeitos dos fármacos , Osteócitos/efeitos dos fármacos , 11-beta-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 11-beta-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 8/genética , Caspase 8/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Regulação da Expressão Gênica , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteócitos/citologia , Osteócitos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Receptor fas/genética , Receptor fas/metabolismoRESUMO
Neuronal cells are highly sensitive to hypoxia and may be subjected to apoptosis when exposed to hypoxia. Several apoptosis-related genes and miRNAs involve in hypoxia-induced apoptosis. This study aimed to examine the role of HIF1α-miR-204-BCL-2 pathway in hypoxia-induced apoptosis in neuronal cells. Annexin V/propidium iodide assay was performed to analyze cell apoptosis in AGE1.HN and PC12 cells under hypoxic or normoxic conditions. The expression of BCL-2 and miR-204 were determined by Western blot and qRT-PCR. The effects of miR-204 overexpression or knockdown on the expression of BCL-2 were evaluated by luciferase assay and Western blot under hypoxic or normoxic conditions. HIF-1α inhibitor YC-1 and siHIF-1α were employed to determine the effect of HIF-1α on the up-regulation of miR-204 and down-regulation of BCL-2 induced by hypoxia. Apoptosis assay showed the presence of apoptosis induced by hypoxia in neuronal cells. Moreover, we found that hypoxia significantly down-regulated the expression of BCL-2, and increased the mRNA level of miR-204 in neuronal cells than that in control. Bioinformatic analysis and luciferase reporter assay demonstrated that miR-204 directly targeted and regulated the expression of BCL-2. Specifically, the expression of BCL-2 was inhibited by miR-204 mimic and enhanced by miR-204 inhibitor. Furthermore, we detected that hypoxia induced cell apoptosis via HIF-1α/miR-204/BCL-2 in neuronal cells. This study demonstrated that HIF-1α-miR-204-BCL-2 pathway contributed to apoptosis of neuronal cells induced by hypoxia, which could potentially be exploited to prevent spinal cord ischemia-reperfusion injury.