miR-218通过靶向TNFR1抑制破骨细胞分化并预防骨质疏松症

miR-218 inhibits osteoclast differentiation and prevents osteoporosis by targeting TNFR1

目的探讨miR-218通过靶向TNFR1在破骨细胞分化中的影响及作用机制。方法采用双侧卵巢切除法制备骨质疏松症模型并进行验证,qRT-PCR检测骨质疏松症小鼠的骨髓单核细胞(BMMs)中miR-218的表达水平;qRT-PCR检测正常小鼠的BMMs在破骨细胞分化过程中NFATc1、TRAF6、miR-218的表达变化,并用TRAP染色观察破骨细胞的生成情况;miR-218模拟物(miR-218 mimics)和miR-218抑制剂(miR-218 inhibitor)转染入BMMs中,qRT-PCR检测NFATc1、TRAF6、miR-218的表达水平,并用TRAP染色观察破骨细胞的生成情况;生物信息学分析和荧光素酶报告基因分析,预测和鉴定miR-218靶标;Western blot检测TNFR1蛋白的表达。结果骨质疏松症小鼠的BMMs中miR-218的表达水平较低(P<0.01);当正常小鼠的BMMs分化为破骨细胞时,NFATc1、TRAF6的表达水平上调(P<0.01),miR-218的表达水平下调(P<0.01),TRAP染色阳性的多核巨细胞形成增加;上调miR-218抑制BMMs在体外分化为破骨细胞,且NFATc1、TRAF6的表达减弱(P<0.01,P<0.05),TRAP染色阳性的多核巨细胞形成减少;通过生物信息学和荧光素酶报告基因检测分析,TNFR1被证实是miR-218的直接靶标,上调miR-218时,TNFR1蛋白表达减少。结论miR-218通过靶向TNFR1在破骨细胞形成中具有重要作用。因此,miR-218在骨质疏松症的治疗中具有治疗潜力。

Objective To explore the effect and mechanism of miR-218 on osteoclast differentiation through targeting TNFR1.Methods The expression of miR-218 in bone marrow mononuclear cells(bmms)was detected by QRT PCR.QRT PCR was used to detect the expression of NFATc1,TRAF6 and miR-218 in bmms of normal mice during osteoclast differentiation,and trap staining was used to observe the osteoclast formation.miR-218 mimics and miR-218 inhibitor were transfected into BMMS.The expression levels of NFATc1,TRAF6 and miR-218 were detected by QRT PCR.The formation of osteoclasts was observed by trap staining.Bioinformatics analysis and luciferase reporter gene analysis were used to predict and identify miR-218 targets.The expression of TNFR1 protein was detected by Western blot.Results The expression level of miR-218 was lower in bmms of osteoporosis mice(P<0.01).When BMMS of normal mice differentiated into osteoclasts,the expression levels of NFATc1 and TRAF6 were up-regulated(P<0.01),the expression level of miR-218 was down regulated(P<0.01),and the formation of trap positive multinucleated giant cells was increased.Up regulation of miR-218 inhibited the differentiation of BMMS into osteoclasts in vitro,decreased the expression of NFATc1 and TRAF6(P<0.01,P<0.05),and decreased the formation of trap positive multinucleated giant cells.Through bioinformatics and luciferase reporter gene analysis,TNFR1 was confirmed to be the direct target of miR-218.When miR-218 was up-regulated,the expression of TNFR1 protein decreased.Conclusion miR-218 plays an important role in osteoclast formation by targeting TNFR1.