解剖学报

解剖学报 ›› 2024, Vol. 55 ›› Issue (6): 708-714.doi: 10.16098/j.issn.0529-1356.2024.06.008

• 细胞和分子生物学 • 上一篇    下一篇

微小RNA-181b-5p通过Sprouty 4蛋白调控骨髓间充质干细胞成骨分化的机制

李娜1 李涛1 姚媛1 李静2* 庄乾宇3* 
  

  1. 1.陕西中医药大学基础医学院第二临床医学院，陕西 咸阳 712046; 2.中国医学科学院基础医学研究所北京协和 医学院基础学院组织工程中心，北京 100005; 3.中国医学科学院北京协和医学院北京协和医院骨科，北京 100730
  • 收稿日期:2023-09-11 修回日期:2023-10-19 出版日期:2024-12-06 发布日期:2024-12-06
  • 通讯作者: 李静;庄乾宇 E-mail:lijing888@ibms.pumc.edu.cn
  • 基金资助:
    四神丸通过调节骨代谢平衡防治慢性胰腺炎性骨质疏松症

Mechanism of microRNA-181b-5p regulating osteogenic differentiation of bone marrow mesenchymal stem cells through Sprouty 4

LI  Na1  LI  Tao1 YAO  Yuan1  LI  Jing2*  ZHUANG  Qian-yu3*    

  1. 1.College of Basic Medicine, the Second Clinical Medical College, Shaanxi University of Chinese Medicine, Shanxi Xian Yang 712046, China; 2.Center of Excellence in Tissue Engineering，Institute of Basic Medical Sciences CAMS, School of Basic Medicine PUMC, Beijing 100005, China;  3.Department of Orthopedics，Peking Union Medical College Hospital，CAMS & PUMC, Beijing 100730, China
  • Received:2023-09-11 Revised:2023-10-19 Online:2024-12-06 Published:2024-12-06
  • Contact: LI Jing;ZHUANG Qian-yu E-mail:lijing888@ibms.pumc.edu.cn

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摘要：

目的  进一步探讨微小RNA(miR)-181b-5p抑制人骨髓间充质干细胞（BMMSCs）成骨分化的调控机制。 方法  取5名健康成人骨髓，分离培养和鉴定人BMMSCs。通过生物信息学网站预测、双荧光素酶报告基因检测、Real-time PCR和Western blotting实验，探究miR-181b-5p与Sprouty4(SPRY4)蛋白的靶向关系。将BMMSCs分为3组：miR-181b-5p过表达阴性对照组；miR-181b-5p过表达组；miR-181b-5p过表达+SPRY4沉默组。碱性磷酸酶（ALP）染色和ALP活性分析法鉴定早期成骨分化效果；茜素红染色检测钙结节沉积情况；Real-time PCR和Western blotting检测成骨分化标志基因的mRNA和蛋白表达水平。 结果  成功分离并鉴定BMMSCs；miR-181b-5p与SPRY4基因mRNA的3’UTR特异性结合，过表达miR-181b-5p后，SPRY4蛋白水平表达显著下调，但在mRNA水平无明显改变，敲降SPRY4解除内源性miR-181b-5p减少对BMMSCs成骨分化的促进作用。 结论  MiR-181b-5p通过下调SPRY4蛋白抑制BMMSCs成骨分化。 

关键词: 微小RNA-181b-5p, Sprouty 4, 骨髓间充质干细胞, 成骨分化, 实时定量聚合酶链反应, 人 

Abstract:

Objective  On the basis of preliminary evidence that microRNA(miR)-181b-5p inhibits osteogenic differentiation of human bone marrow mesenchymal stem cells (BMMSCs), the regulatory mechanism was further explored. Methods  Isolation, culture and identification of BMMSCs from the bone marrow of five healthy adults. The targeting relationship between miR-181b-5p and Sprouty 4(SPRY4) was investigated by bioinformatics software prediction, double luciferase reporter gene detection, Real-time PCR and Western blotting experiments. BMMSCs were divided into three groups, miR-181b-5p overexpression negative control group; miR-181b-5p overexpression group; miR-181b-5p overexpression +SPRY4 silenced group. Alkaline phosphatase(ALP) staining and ALP activity analysis were used to determine the effect of early osteogenic differentiation. The precipitation of calcium nodules was detected by alizarin red staining. The mRNA and protein expression levels of osteogenic differentiation marker genes were detected by Real-time PCR and Western blotting.  Results  BMMSCs were successfully isolated and identified. MiR-181b-5p specifically binds to the 3’UTR of SPRY4 mRNA. After overexpression of miR-181b-5p, the expression of SPRY4 protein level was significantly down-regulated, but there was no significant change in mRNA level. Knocking down the target gene SPRY4 blocked the effect of miR-181b-5p inhibitors on promoting osteogenic differentiation of cells. Conclusion  MiR-181b-5p inhibits osteogenic differentiation of BMMSCs by downregulating SPRY4 protein. 

Key words: MicroRNA-181b-5p, Sprouty 4, Bone marrow mesenchymal stem cell, Osteogenic differentiation, Rea, -time PCR, Human

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