miR-125a/TRAF6调控通路在破骨细胞分化中的作用研究

Effect of miR-125a/TRAF6/NFATC1 loop mediates on osteoclastogenesis

目的研究miR-125a在破骨细胞分化过程中的作用及其作用机制,方法单核细胞集落刺激因子(M-CSF)和核因子-κB配体的受体激活剂(RANKL)诱导CD_(14)^+PBMCs向破骨细胞分化。生物信息学运算预测miR-125a的靶基因以及结合于miR-125a启动子区域的转录因子。实时定量PCR法检测miR-125a以及其他相关基因的表达。过表达实验和抑制试验用于研究miR-125a在破骨细胞分化中的作用及其与靶基因之间的相互关系。荧光素酶报告基因实验验证miR-125a与其靶基因之间的结合。结果(1)miR-125a表达在CD_(14)^+PBMCs前体细胞高表达,随着诱导时间的延续逐渐下降,在诱导第5天开始明显下降并在第15天达到最低值,表明miR-125a的表达在破骨细胞分化过程中呈现明显下降趋势。(2)miR-125a参与调控RANKL和M-CSF诱导的破骨细胞分化,过表达miR-125a明显抑制了TRAP和NFATc1的mRNA表达和CD_(14)^+PBMCs向破骨细胞的分化。(3)miR-125a直接作用于靶基因肿瘤坏死因子相关因子6(TRAF6),TRAF6是RANKL/RANK/NFATc1信号转导通路的转录因子,过表达miR-125a降低了TRAF6的蛋白表达水平,而TRAF6的mRNA水平无明显改变。结论 miR-125a通过作用于新的FRAF6/NFATC1/miR-125a负反馈调控环路在破骨细胞的分化中发挥了重要的调控作用,调控miR-125a的表达可能成为骨代谢疾病新的治疗靶点。

Objective To investigate the effect of miR-125 a in the process of osteoclast.Methods Monocyte colony stimulating factor （M-CSF） and nuclear factor kappa B ligand /receptor activator （RANKL） induced CD1＋4 PBMCs to osteo-clas.The target gene bioinformatics arithmetic predictive miR-125a and miR-125a binding to initiate transcription factor sub region.To detect the expression of miR-125 a using real-time quantitative PCR and other related genes.Overexpression experi-ments and inhibition test were used to investigate the relationship between effect of miR -125 a in osteoclast differentiation and target gene.Used the luciferase reporter gene experiments to verify the combination between miR -125 a and its target gene.Re-sults （1） miR-125a expression in CD1＋4 PBMCs precursor cells showed high expression level , with a continuation of the in-duction time , the expression decreased gradually , and reached the lowest value at fifteenth days , which showed that the ex-pression of miR-125a revealed a clear downward trend in osteoclast differentiation process.（2） miR-125a are involved in the regulation of RANKL and M /CSF induced osteoclast differentiation , over expression of miR-125 a significantly inhibited the expression of mRNA and CD1＋4 PBMCs TRAP and NFATc1 to differentiate into osteoclast.（3） miR-125a direct effect on tar-get gene expression of tumor necrosis factor related factor 6 （TRAF6）, TRAF6 is a transcription factor of the RANKL/RANK/NFATc1 signaling pathway, overexpression of miR-125a decreased the protein expression level of TRAF 6, but no significant change of TRAF6 mRNA level were found.Conclusion It proved that the miR-125 a played an important role in the regulation of osteoclast differentiation by acting on the new TRAF 6/NFATC1/miR-125a negative feedback control loop , expression and regulation of miR-125a may become a new therapeutic target for metabolic bone diseases.