miR-187-5p通过RANKL/NFκB信号通路抑制骨质疏松症中的破骨细胞活性

miR-187-5p alleviates osteoporosis through inhibiting activity of osteoclast via RANKL/NFκB

目的探讨miR-187-5p通过RANKL/NFκB信号通路对骨质疏松症中破骨细胞活性的影响及机制分析。方法使用小鼠巨噬细胞264.7细胞株(RAW246.7),设置正常组(con)、核因子κB配体的受体激活子(receptor activator of nuclear factor-κBligand,RANKL)诱导的骨质疏松症组(RANKL-induced OP)、阴性对照(mimic negative control,mimic NC)、OP+miR-187-5p模拟物(OP+miR-187-5p mimic),通过细胞计数试剂盒(the cell counting kit 8,CCK8)检测破骨细胞的增殖水平,RT-qPCR检测TRAPmRNA、miR-187-5p表达水平,免疫印迹检测p65和TNFα蛋白表达。最后,通过双荧光素酶报告基因(dual luciferase reporter assays)检测miR-187-5p和p65间的相互作用。结果RAW264.7细胞转染miR-187-5p后,OP组的细胞增殖水平和TRAP mRNA水平显著高于con组(P=0.008、0.017),OP+miR-187-5p mimic组细胞增殖水平和TRAP mRNA水平显著低于OP+miR-187-5p NC组(P=0.023、0.037)。而miR-187-5p在OP组显著低于con组(P=0.031),转染miR-187-5p mimic后升高其表达水平(P=0.041)。此外,OP组的p65和TNFα蛋白水平明显高于con组(P=0.034;P=0.024),OP+miR-187-5p mimic组的p65和TNFα蛋白水平显著低于OP+miR-187-5p NC组(P=0.028;P=0.036)。同时OP组的p65显著高于con组(P=0.039),OP+miR-187-5p mimic组的p65基因水平明显高于OP+miR-187-5p NC(P=0.025)。双荧光素酶报告分析,miR-187-5p与p65间存在靶向关系。结论miR-187-5p通过抑制NFκB信号通路抑制破骨细胞的活性。

Objective To investigate the role of microRNA-187-5p(miR-187-5p)on osteoporosis(OP)as well as the related molecular mechanism.Methods The level of miR-187-5p and tartrate-resistant acid phosphatase(TRAP,the osteoclast marker)was quantitatively detected in TRANKL-induced OP group and control(con)group by reverse transcription-quantitative PCR(RT-qPCR);Cell counting kit 8(CCK8)was used to measure the viability of murine macrophage cell line(RAW264.7);Western blot was used to detect the expression of p65 and TNFα.Moreover,the dual luciferase reporter assays was applied to detect the interaction between miR-187-5p and p65.Results The osteoclast proliferation determined by CCK8 and the mRNA level of TRAP detected by RT-qPCR demonstrated that the growth of osteoclast was inhibited in OP group compared with that in con group(P=0.008;P=0.017),which reversed by miR-187-5p mimic stimulation(P=0.023;P=0.037).The miR-187-5p was lowly expressed in OP group compared with that in con group(P=0.031),which was increased by miR-187-5p mimic treatment compared with miR-187-5p NC treatment(P=0.041).Moreover,western blot indicated that the protein level of p65 and TNFαin OP group were up-regulated compared with that in con group(P=0.034;P=0.024),which restored by the miR-187-5p mimic co-incubation(P=0.028;P=0.036).Moreover,the RT-qPCR indicated that the mRNA level of p65 in OP group was also increased compared with that in con group(P=0.039),and the miR-187-5p mimic co-incubation decreased the mRNA level of p65,compared with miR-187-5p NC group(P=0.025).The dual luciferase reporter assays indicated that there was an interaction between miR-187-5p and p65.Conclusion miR-187-5p alleviates OP through inhibiting the activity of osteoclast via RANKL/NFκB signaling.