柚皮苷调控miR-199a-5p/ECE1分子轴促进骨损伤修复

Naringin Promotes the Repair of Bone Injury by Regulating the Axis of miR-199a-5p/ECE1

目的探讨柚皮苷通过调控mi R-199a-5p/ECE1分子轴对骨损伤修复的影响。方法 Western blotting检测ECE1及成骨分化相关标志物的表达水平;RT-qPCR检测mi RNAs相对表达水平;双荧光素酶报告基因验证mi R-199a-5p和ECE1靶向关系;MTT检测MC3T3-E1细胞增殖活力;碱性磷酸酶(ALP)检测MC3T3-E1细胞成骨分化能力;茜素红染色检测MC3T3-E1细胞矿化情况。结果柚皮苷处理显著促进MC3T3-E1细胞增殖活力、成骨分化指标Runx2、OPN、BMPs、OC等的表达、ALP活性以及细胞钙化能力水平(P<0.01)。其中20ng/m L浓度处理的效果最优。RT-qPCR检测结果显示,柚皮苷能显著上调mi R-199a-5p的表达水平。双荧光素酶报告基因检测结果表明ECE1是mi R-199a-5p的一个靶基因;并且,mi R-199a-5p靶向下调ECE1的表达。进一步实验表明,敲降mi R-199a-5p显著降低柚皮苷对MC3T3-E1细胞增殖活力、ALP活性和钙矿化的促进作用(P<0.01);敲降ECE1显著促进了柚皮苷对MC3T3-E1细胞增殖活力、ALP活性和生物矿化能力的促进作用(P<0.01);柚皮苷处理加同时敲降mi R-199a-5p和ECE1组MC3T3-E1细胞增殖活力、ALP活性和钙矿化能力与柚皮苷组无显著差异(P>0.05)。对细胞成骨分化标志的检测也证明了上述结果。由此可知,柚皮苷通过上调mi R-199a-5p对ECE1靶向下调作用,进而促进MC3T3-E1细胞增殖活力、成骨分化和矿化能力。结论柚皮苷处理显著促进了MC3T3-E1细胞的成骨分化水平;这种作用是通过上调mi R-199a-5p/ECE1分子轴实现的。

Objective To investigate the effect of naringin on bone injury repair by regulating the axis of miR-199a-5p/ECE1.Methods The expression levels of ECE1 and osteogenic differentiation related markers were detected by Western blotting.RT-qPCR was used to detect the relative expression level of miRNAs.The targeting relationship between miR-199a-5p and ECE1 was verified by dual-luciferase reporter gene.The proliferation activity of MC3T3-E1cells was detected by MTT.The osteogenic differentiation of MC3T3-E1cells was determined by alkaline phosphatase(ALP).Mineralization of MC3T3-E1 cells was detected by alizarin red staining.Results Naringin treatment significantly promoted the proliferation activity of MC3T3-E1cells,the expressions of osteogenic differentiation indicators Runx2,OPN,BMPs,OC,ALP activity and the level of calcification.Among them,the effect of 20 ng/mL concentration treatment was the best.The results of RT-qPCR showed that naringin could significantly up-regulate the expression level of miR-199a-5p.The results of double luciferase reporter gene showed that ECE1 was a target gene of miR-199a-5p.Furthermore,miR-199a-5p targeted to down-regulate the expression of ECE1.Further experiments showed that miR-199a-5p knockdown significantly reduced the promoting effect of naringin on MC3T3-E1cell proliferation activity,ALP activity and calcium mineralization.Knockdown of ECE1 significantly promoted the proliferation activity,ALP activity and biomineralization ability of MC3T3-E1cells by naringin.The proliferation activity,ALP activity and calcium mineralization ability of MC3T3-E1cells in miR-199a-5p and ECE1 groups were not significantly different from those in the naringin group.These results were also supported by the detection of osteogenic differentiation markers.Therefore,naringin can down-regulate ECE1 by up-regulating miR-199a-5p,thus promoting proliferation,osteogenic differentiation and mineralization of MC3T3-E1cells.Conclusion Naringin treatment significantly promoted the osteogenic differentiation of MC3T3-E1cells.This effect is achieved by up-regulating the molecular axis of miR-199a-5p/ECE1.