miR-206靶向Cx43调控ERK1/2通路在兔激素性股骨头坏死中的作用

The role of miR-206 on regulation of ERK1/2 signaling pathway by targeting Cx43 in rabbit steroid-induced avascular necrosis of femoral head model

目的探讨微小RNA(MicroRNA,miR)-206调控缝隙连接蛋白(connexin,Cx)43激活蛋白激酶(ERK)1/2通路是否参与兔激素性股骨头坏死的发生发展,并初步阐明其机制。方法成年新西兰大白兔60只,随机分为实验组和对照组各30只,实验组接受内毒素(10 mg/kg)联合甲强龙(20 mg/kg)注射制作激素性股骨头坏死模型。注射2周、8周和16周后两组动物行MRI检查,HE染色确定模型建立成功;模型兔与相应对照组股骨头标本行原位杂交和实时荧光定量PCR(qPCR)检测miR-206表达,Western blot、免疫组织化学和qPCR检测Cx43、ERK1/2和Runx2基因及蛋白表达。结果建模成功率为70%;原位杂交显示miR-206表达定位于兔股骨头髓腔、成骨细胞及少量骨细胞。造模后2周、8周和16周,与对照组比较,实验组兔股骨头内miR-206表达上调;Cx43、Runx2基因表达下调,Cx43、ERK1/2、Runx2蛋白表达下调。结论miR-206可通过下调其靶蛋白Cx43,抑制ERK1/2信号通路,抑制成骨分化,参与激素性股骨头坏死发生发展及修复。

Objective To explore the role of MicroRNA(miR)-206 on regulating of extracellular signal-regulated protein kinase(ERK)1/2 signaling pathway by targeting connexin(Cx)43 in rabbit model of steroid-induced avascular necrosis of femoral head.Methods Sixty mature rabbits were randomly divided into a model group(n=30)and a control group(n=30).Rabbit model of femoral head necrosis was made with lipopolysaccharide(LPS)and methylprednisolone(MPS).The establishment of the model was determined by MRI and HE staining after modeled 2、8 and 16 weeks.Total RNA and protein were extracted from the femoral head.In situ hybridization and Quantitative Real-time PCR(qPCR)were employed to detect the change of miRNA-206;qPCR,western blot and immunohistochemistry were used to detect the expression of Cx43、ERK1/2 and Runx2.Results The model success rate was 70%.In situ hybridization result showed that the miR-206 expressed in rabbit femoral head medullary space,osteoblasts and osteocyte.Compared with the control group,miR-206 expression of the model group up-regulated at 2 weeks,8 weeks and 16 weeks after modeling,the expression of Cx43 and Runx2 mRNA in the model group down-regulated,the expression of Cx43、ERK1/2 and Runx2 protein in the model group also down-regulated.Conclusions miR-206 can be involved in the occurrence,development and repair of steroid-induced femoral head necrosis by downregulating its target protein CX43,inhibiting the ERK1/2 signaling pathway and osteogenic differentiation.