破骨细胞分化中NFATc1/miR-125a作用通路的研究

NFATc1/miR-125a function pathway mediates osteoclastogenesis

目的研究破骨细胞分化中miR-125a受转录因子NFATc1的调控模式及其作用机制,寻找骨代谢疾病新的治疗靶点。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)^+外周血单核细胞(PBMCs)向破骨细胞分化。生物信息学运算预测结合于miR-125a启动子区域的转录因子,过表达实验和抑制实验用于研究转录因子对miR-125a在破骨细胞分化中表达的影响,荧光素酶报告基因实验验证miR-125a与其靶基因之间的结合,凝胶迁移电泳实验(EMSA)和染色质免疫沉淀实验(CHIP)验证转录因子与miR-125a启动子区域的结合。结果凝胶迁移电泳和染色质免疫沉淀实验证明了NFATc1结合于miR-125a的启动子靶位点。过表达NFATc1抑制miR-125a的表达;抑制NFATc1的表达则升高miR-125a的表达。结论 miR-125a在作用于其靶基因——肿瘤坏死因子受体相关因子(TRAF)6的同时受到转录因子NFATc1的负反馈调控,发挥其在破骨细胞分化中的调控作用。表明通过调控NFATc1的表达来改变miR-125a的表达,可能成为骨代谢疾病新的治疗靶点。

Objective To investigate the mode and mechanism of breaking the regulation of bone cell differentiation in miR-125 a by the transcription factor NFATcl,and to find a new therapeutic target for metabolic bone diseases.Methods Monocyte colony stimulating factor（M-CSF） and nuclear factor kappa B ligand receptor activator（RANKL） blood monocytes induced CD14+ peripheral（PBMCs） were differentiated to the osteoclast.Combined with biological information from miR-125 a to start transcription factor promoter region to learn operational forecasting,over expression experiments and inhibition experiments were used to investigate the effect of miR on expression of transcription factor 125 a in osteoclast differentiation,the combination between the luciferase reporter gene experiments to verify the miR-125 a and its target gene,gel mobility shift electrophoresis experiment（EMSA） and chromatin immunoprecipitation（CHIP） experiments verify the transcription factor and miR-125 a binds promoter region.Results The gel mobility shift electrophoresis and chromatin immunoprecipitation experiments proved that the target sites of NFATcl promoter binding to miR-125 a.Overexpression of NFATcl inhibits expression of miR-125a;the expression of inhibiting the expression of NFATcl was increased the miR-125 a.Conclusion When miR-125 a worked on the target genes- tumor necrosis factor receptor associated factor（TRAF） 6 at the same time by negative feedback regulation of the transcription factor NFATcl,its play the role in the regulation of osteoclast differentiation.It is demonstrated that the expression of NFATcl by regulating the expression of miR-125 a,it may become a new therapeutic target for metabolic bone diseases.