miR-106b介导RANKL/RANK/OPG通路抗骨质疏松症的实验研究

Experimental Study of miR-106b Mediated RANKL/RANK/OPGPathway Against Osteoporosis

目的:探讨微小核糖核酸106b(miR-106b)介导核因子κB受体激活因子配体(RANKL)/核因子κB受体激活因子(RANK)/骨保护素(OPG)通路抗骨质疏松症的作用。方法:取60只Wistar大鼠切除卵巢制备骨质疏松症大鼠模型,将建模成功的51只大鼠随机分为模型组、miR-106b mimics-NC组、miR-106b mimics组、miR-106b inhibitor-NC组、miR-106b inhibitor组,除模型组外其余各组分别转染miR-106b模拟物阴性对照、miR-106b模拟物、miR-106b抑制剂阴性对照和miR-106b抑制剂,另取10只大鼠仅切除卵巢附近脂肪作为假手术组。X线骨密度仪检测大鼠股骨骨密度;苏木素-伊红(HE)染色观察大鼠右侧股骨组织病理学变化;实时荧光定量聚合酶链式反应(RT-qPCR)、免疫印迹(WB)分别检测大鼠右侧股骨组织miR-106b表达、RANKL、RANK、OPG信使核糖核酸(mRNA)表达和蛋白的表达;双荧光素酶报告基因实验验证miR-106b和RANKL的靶向关系。结果:与模型组、miR-106b mimics-NC组比,miR-106b mimics组右侧股骨组织骨密度、miR-106b表达、OPG mRNA和蛋白表达升高(P<0.05),RNAKL mRNA和蛋白表达降低(P<0.05);与模型组、miR-106b inhibitor-NC组比,miR-106b inhibitor组右侧股骨组织骨密度、miR-106b表达、OPG mRNA和蛋白表达均降低(P<0.05),RNAKL mRNA和蛋白表达升高(P<0.05);假手术组大量骨小梁排列紧密、厚度均匀;模型组、miR-106b mimics-NC组、miR-106b inhibitor-NC组骨细胞数减少,排列紊乱,骨小梁间隙大;miR-106b mimics组骨细胞数增多,排列紧密,骨小梁厚度增加;miR-106b inhibitor组骨细胞数缺失,排列紊乱,骨小梁间隙较大。双荧光素酶报告基因实验证实miR-106b靶向调控RANKL。结论:上调miR-106b表达可抑制RANKL表达,促进OPG表达,增加骨质疏松症大鼠股骨骨密度,减轻右侧股骨组织病理损伤。

Objective:To explore the anti-osteoporosis effect of mirco ribonucleic acid 106b(miR-106b)mediated activator receptor of nuclear factorκB ligand(RANKL)/receptor activator of nuclear factorκB(RANK)/osteoprotegerin(OPG)pathway.Methods:Sixty Wistar rats were ovariectomized to prepare the osteoporosis rat model,and another 10 rats were only removed the fat around the ovary as the sham operation group.The 51 rats successfully modeled were randomly divided into model group,miR-106b mimics-NC group,miR-106b mimics group,miR-106b inhibitor-NC group and miR-106b inhibitor group.All groups except the model group were transfected with miR-106b mimic negative control,miR-106b mimic,miR-106b inhibitor negative control and miR-106b inhibitor respectively,and 10 rats were removed only the fat near the ovary as the sham operation group.X-ray bone densitometry was used to measure the bone mineral density of the rat femur;hematoxylin-eosin(HE)staining was used to observe the histopathological changes of the rat right femur;real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and immunoblotting(WB)were used to detect miR-106b expression,RANKL,RANK,OPG messenger ribonucleic acid(mRNA)expression and protein expression of the rat right femur respectively;dual luciferase reporter gene assay was performed to verify the targeting relationship between miR-106b and RANKL.Results:Bone mineral density,miR-106b expression,OPG mRNA and protein expression were increased(P<0.05),RNAKL mRNA and protein expression were decreased(P<0.05)in the right femoral tissue of the miR-106b mimics group compared to the model group and miR-106b mimics-NC group.Compared with the model group and miR-106b inhibitor-NC group,BMD,miR-106b expression,OPG mRNA and protein expression in the right femoral tissue of the miR-106b inhibitor group were reduced(P<0.05)and RNAKL mRNA and protein expression were increased(P<0.05).In the sham-operated group,a large number of bone trabeculae were closely arranged and of uniform thickness;in the model group,miR-106b mimics-NC group and miR-106b inhibitor-NC group,the number of bone cells was reduced,the arrangement was disordered and the gap between bone trabeculae was large;in the miR-106b mimics group,the number of bone cells was increased,the arrangement was tight and the thickness of bone trabeculae was increased;in the miR-106b inhibitor group showed a lack of osteoblasts,disorganized arrangement and large trabecular gaps.The dual luciferase reporter gene assay confirmed that miR-106b targets and regulates RANKL.Conclusion:Up-regulation of miR-106b expression inhibited RANKL expression,promoted OPG expression,increased femoral bone mineral density and reduced histopathological damage to the right femur in osteoporotic rats.