微小RNA-129-5p通过成纤维细胞生长因子2调节老年性骨质疏松性骨折中骨分化和骨稳态的功能研究

Function of microRNA-129-5p in regulating bone differentiation and bone homeostasis in senile osteoporotic fractures through fibroblast growth factor 2

目的:探讨破骨细胞分化过程中微小RNA(miR)-129-5p的失调以及miR-129-5p在破骨细胞分化中的确切作用机制。方法:首先建立核因子-κB受体活化因子配体(RANKL)诱导鼠源单核巨噬细胞(RAW264.7)分化的破骨细胞,实时荧光定量聚合酶链反应(RT-qPCR)检测miR-129-5p在破骨细胞分化前后的表达差异。然后用miR-129-5p模拟物处理RANKL诱导的RAW264.7细胞,验证miR-129-5p对破骨细胞分化的影响。细胞计数试剂盒(CCK-8)实验用于检测破骨细胞的增殖活性,抗酒石酸酸性磷酸酶染色确定破骨细胞数目,蛋白质印迹法检测破骨相关标志物的蛋白表达。最后,通过RT-qPCR或蛋白质印迹法分析miR-129-5p对成纤维细胞生长因子2(FGF2)表达的影响。组间统计学差异采用T-Test法检验。结果:miR-129-5p在RANKL组的表达水平低于对照组(0.405±0.007比0.984±0.031,t=24.070,P<0.01)。在功能上,上调miR-129-5p可以抑制破骨细胞的分化,CCK-8实验结果显示,miR-129-5p处理组的破骨细胞活力低于阴性对照组(1.096±0.027比0.549±0.006,t=33.670,P<0.001)。机制上,miR-129-5p对破骨细胞分化的抑制作用,可能与负调节FGF2表达有关。RT-qPCR实验结果显示,miR-129-5p处理组破骨细胞中FGF2 mRNA表达水平明显低于对照组(1.001±0.026比0.151±0.004,t=48.001,P<0.01)。此外,功能挽救实验结果表明过表达FGF2可以抵消miR-129-5p上调对破骨细胞分化的抑制作用。RT-qPCR实验结果显示,miR-129-5p mimic+OE-FGF2组破骨细胞中FGF2的水平高于miR-129-5p mimic+OE-NC组(0.973±0.059比5.108±0.018,t=86.059,P<0.001),差异均有统计学意义。结论:miR-129-5p通过负调节FGF2表达,显著抑制了破骨细胞分化。

Objective To investigate the dysregulation of miR-129-5p during osteoclast differentiation and the exact mechanism of miR-129-5p in osteoclast differentiation.Methods Firstly,osteoclasts induced by receptor activator of nuclear factor-κB ligand(RANKL)into differentiation of murine mononuclear macrophages(RAW264.7)were established,and the expression of miR-129-5p before and after osteoclast differentiation was detected by RT-qPCR.Then,RAW264.7 cells induced by RANKL were treated with miR-129-5p simulators to verify the effect of miR-129-5p on osteoclast differentiation.The proliferative activity of osteoclasts was detected by cell counting kit-8(CCK-8)assay,the number of osteoclasts was determined by tartrate-resistant acid phosphatase staining,and the protein expression of osteoclast-related markers was detected by Western blotting.Finally,the effect of miR-129-5p on fibroblast growth factor 2(FGF2)expression was analyzed by RT-qPCR or Western blotting.Results The expression level of miR-129-5p in RANKL group was lower than that in control group(0.405±0.007 vs.0.984±0.031,t=24.070,P<0.01).In terms of function,upregulation of miR-129-5p can inhibit osteoclast differentiation.The results of CCK-8 experiment showed that compared with the negative control group,the activity of osteoclasts was decreased after the intervention of miR-129-5p mimics(1.096±0.027 vs.0.549±0.006,t=33.670,P<0.001).The inhibitory effect of miR-129-5p on osteoclast differentiation may be related to the negative regulation of FGF2 expression.The results of RT-qPCR showed that compared with the negative control group,the expression of FGF2 mRNA in osteoclasts after the intervention of miR-129-5p mimics was inhibited(1.001±0.026 vs.0.151±0.004,t=48.001,P<0.01).In addition,functional salvage experiments showed that overexpression of FGF2 could counteract the inhibitory effect of miR-129-5p upregulation on osteoclast differentiation.RT-qPCR results showed that compared with miR-129-5p mimic+OE-NC group,miR-129-5p mimic+OE-FGF2 group increased the level of FGF2 in osteoclasts(0.973±0.059 vs.5.108±0.018,t=86.059,P<0.001),and the differences were significant.Conclusion MiR-129-5p inhibits osteoclast differentiation by negatively regulating FGF2 expression.