MiR-2861/TIMP4轴在地塞米松-诱导小鼠骨质疏松症模型的分子机制研究  

Molecular mechanism of miR-2861/TIMP4 axis in mice with glucocorticoid-induced osteoporosis

作　　者：孟开顺 洪路贤 梁莉萍 MENG Kaishun;HONG Luxian;LIANG Liping(Department of Geriatrics,People’s Hospital of Sanya,Sanya 572000,China)

机构地区：[1]三亚市人民医院老年病科,海南三亚572000

出　　处：《中国骨质疏松杂志》2023年第3期336-342,360,共8页Chinese Journal of Osteoporosis

基　　金：海南省卫生健康行业科研项目(21A200293)。

摘　　要：目的研究与地塞米松-诱导的骨质疏松症密切相关的靶microRNAs分子及其作用机制。方法在体动物实验,将小鼠随机分为Control组和地塞米松-诱导的骨质疏松症小鼠(Model)组;microRNA表达谱测序分析上述两组小鼠股骨近端骨组织中microRNAs的显著变化;生物信息学分析miR-2861与TIMP4的序列互作位点;qPCR检测两组小鼠股骨组织中miR-2816和TIMP4的RNA表达情况。双荧光素酶报告基因检测确证miR-2861与TIMP4的序列互作。体外细胞实验,对细胞进行地塞米松(Dex)处理后,分别敲低miR-2861或TIMP4;Annexin V-PI双标记流式细胞术检测各种处理前后细胞凋亡情况,Western blot检测各种处理前后凋亡相关蛋白的表达情况。结果microRNA表达谱测序结果显示,与Control组小鼠相比,Model组小鼠股骨近端骨组织中miR-2861显著上调。双荧光素酶报告基因检测确证miR-2861对TIMP4的负调控作用。qPCR检测确证与Control组小鼠相比,Model组小鼠股骨组织中miR-2816表达显著上调,TIMP4表达显著下调(P<0.05)。体外细胞实验,与Control组相比,Dex处理组细胞凋亡显著上升(P<0.05),凋亡相关蛋白Cleaved-Caspase-3、Bax/Bcl-2显著升高(P<0.05);与Dex+inhibitor NT组相比,Dex+miR-2861 inhibitor组细胞凋亡显著降低,凋亡相关蛋白表达显著下调,TIMP4表达显著升高(P<0.05);与Dex+TIMP4 siRNA NT组相比,Dex+TIMP4 siRNA组细胞凋亡显著升高,凋亡相关蛋白表达显著升高,TIMP4表达显著降低(P<0.05)。结论Dex通过miR-2861/TIMP4轴促进地塞米松-诱导的骨质疏松症小鼠成骨细胞凋亡。Objective To detect the targeted microRNAs closely related to glucocorticoid-induced osteoporosis and reveal their molecular mechanism.Methods In in vivo animal studies,mice were randomly divided into Control group and glucocorticoid-induced osteoporosis mice(Model)group.MicroRNA profiling was used to analyzed the microRNAs changed significantly in the proximal femur bone tissue of the above two groups of mice.Bioinformatics analysis of the interaction sites between miR-2861 and TIMP4 was conducted.RNA expression of miR-2816 and TIMP4 in femur tissue of the two groups of mice were detected with qPCR.Dual-luciferase reporter assay confirmed the interaction between miR-2861 and TIMP4.In in vitro studies,after cells were treated with dexamethasone(Dex),miR-2861 or TIMP4 was knocked down,respectively.Annexin V-PI double-labeled flow cytometry was used to detect cell apoptosis.Western blotting was used to detect the expression of apoptosis-related proteins.Results MicroRNA profiling result showed that miR-2861 was significantly up-regulated in the proximal femur bone tissue in the Model group mice compared with that in the Control group mice.Dual-luciferase reporter assay confirmed the negative regulation of miR-2861 on TIMP4.qPCR assay confirmed that compared with that in the Control group,the expression of miR-2816 in the femur tissue of the Model group mice was significantly up-regulated,and the expression of TIMP4 was significantly down-regulated(P<0.05).In in vitro cell experiments,compared with that in the Control group,the apoptosis of Dex group increased significantly(P<0.05).The apoptosis-related proteins,Cleaved-Caspase-3 and Bax/Bcl-2,increased significantly(P<0.05).Compared with Dex+inhibitor NT group,the apoptosis in Dex+miR-2861 inhibitor group reduced significantly,the expression of apoptosis-related proteins was significantly down-regulated,and the expression of TIMP4 increased significantly(P<0.05).Compared with those in Dex+TIMP4 siRNA NT group,the apoptosis of Dex+TIMP4 siRNA group increased signifi

关 键 词：地塞米松 骨质疏松症 成骨细胞 miR-2816 TIMP4 

分 类 号：R318[医药卫生—生物医学工程]