CAJ | 학술논문

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CD1＋4 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CD1＋4 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-125 a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CD1＋4 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-125 a通过作用于新的TRAF6／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。
목적：연구miR-125a재파골세포분화과정중적작용급기작용궤제，방법단핵세포집락자격인자（M-CSF）화핵인자-κB배체적수체격활제（RANKL）유도CD1＋4 PBMCs향파골세포분화。생물신식학운산예측miR-125a적파기인이급결합우miR-125a계동자구역적전록인자。실시정량PCR법검측miR-125a이급기타상관기인적표체。과표체실험화억제시험용우연구miR-125a재파골세포분화중적작용급기여파기인지간적상호관계。형광소매보고기인실험험증miR-125a여기파기인지간적결합。결과（1） miR-125a표체재CD1＋4 PBMCs전체세포고표체，수착유도시간적연속축점하강，재유도제5천개시명현하강병재제15천체도최저치，표명miR-125 a적표체재파골세포분화과정중정현명현하강추세。（2）miR-125a삼여조공RANKL화M-CSF유도적파골세포분화，과표체miR-125a명현억제료TRAP화NFATc1적mRNA표체화CD1＋4 PBMCs향파골세포적분화。（3）miR-125a직접작용우파기인종류배사인자상관인자6（TRAF6），TRAF6시RANKL／RANK／NFATc1신호전도통로적전록인자，과표체miR-125a강저료TRAF6적단백표체수평，이TRAF6적mRNA수평무명현개변。결론 miR-125 a통과작용우신적TRAF6／NFATC1／miR-125a부반궤조공배로재파골세포적분화중발휘료중요적조공작용，조공miR-125a적표체가능성위골대사질병신적치료파점。
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-clast.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 osteoclasts.(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 .