hsa-miRNA-223-3p调控人骨髓间充质干细胞成骨分化的作用  

Role of hsa-miRNA-223-3p in regulating osteogenic differentiation of human bone marrow mesenchymal stem cells

作　　者：耿瑶 尹志良 李兴平 肖东琴[3] 侯伟光 Geng Yao;Yin Zhiliang;Li Xingping;Xiao Dongqin;Hou Weiguang(Southwest Medical University,Luzhou 646000,Sichuan Province,China;Chengfei Hospital,Chengdu 610000,Sichuan Province,China;Research Institute of Tissue Engineering and Stem Cells,Nanchong Central Hospital,the Second Clinical College of North Sichuan Medical College,Nanchong 637000,Sichuan Province,China;AVIC 363 Hospital,Chengdu 610000,Sichuan Province,China)

机构地区：[1]西南医科大学,四川省泸州市646000 [2]成飞医院,四川省成都市610000 [3]川北医学院第二临床医学院·南充市中心医院组织工程与干细胞研究所,四川省南充市637000 [4]航空工业三六三医院,四川省成都市610000

出　　处：《中国组织工程研究》2021年第7期1008-1013,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金项目(81171472),项目参与人:肖东琴;四川省科技厅应用基础项目(2016JY0123),项目负责人:肖东琴;四川省科技厅应用基础项目(2018JY0100),项目参与人:肖东琴;南充市市校合作科研专项资金(19SXHZ0099,19SXHZ0236),项目参与人:肖东琴。

摘　　要：背景:研究发现miR-223-3p在骨质疏松患者体内高表达,但其相关机制研究甚少。目的:探讨hsa-miR-223-3p对骨髓间充质干细胞成骨分化的调节作用。方法:Percoll密度梯度离心法分离、培养骨髓间充质干细胞,诱导其成骨分化,然后过表达hsa-miR-223-3p,RT-qPCR和Western blot方法检测Wnt信号通路标志物Wnt5a及其下游信号分子OPG、RUNX2的mRNA和蛋白水平变化。构建pmirGLO/Wnt5a-3’UTR和pmirGLO/Wnt5a-3’UTR mut质粒,分别与hsa-miR-223-3p mimics/对照质粒共转染至293T细胞,双荧光素酶报告基因系统验证hsa-miR-223-3p与Wnt5a的靶定关系。结果与结论:①随着成骨诱导时间的延长,细胞培养液中碱性磷酸酶水平逐渐增加,茜素红染色可见暗红色或红褐色钙盐结节沉积逐渐增加;②过表达hsa-miR-223-3p后,细胞中Wnt5a、OPG、RUNX2的mRNA和蛋白水平降低;③hsa-miR-223-3p直接靶定Wnt5a的3’UTR区,降低Wnt5a的荧光素酶活性;突变掉Wnt5a的结合位点,靶定作用消失。封闭hsa-miR-223-3p后,野生型Wnt5a的荧光素酶活性增加,突变型Wnt5a的荧光素酶活性不变;④结果表明,在骨髓间充质干细胞向成骨细胞分化过程中,过表达hsa-miR-223-3p可以影响Wnt信号通路标志物Wnt5a及其下游信号分子OPG、RUNX2的表达。hsa-miR-223-3p可直接与Wnt5a结合,Wnt5a作为hsa-miR-223-3p的下游靶基因,受其负向调控。BACKGROUND:Studies have found that miR-223-3p is highly expressed in osteoporosis patients,but the related mechanism is rarely studied.OBJECTIVE:To investigate the regulatory effect of hsa-miR-223-3p on osteogenic differentiation of bone marrow mesenchymal stem cells.METHODS:Bone marrow mesenchymal stem cells were isolated and obtained by Percoll density gradient centrifugation.The cells were induced into osteogenic differentiation,and overexpressed hsa-miR-223-3p.RT-qPCR and western blot assay were used to detect the changes in the mRNA and protein levels of Wnt signaling pathway markers Wnt5a and its downstream signaling molecules OPG and RUNX2.PmirGLO/Wnt5a-3'UTR and pmirGLO/Wnt5a-3'UTR mut plasmids were constructed and co-transfected with hsa-miR-223-3p mimics/control plasmid into 293T cells to verify the targeting relationship between hsa-miR-223-3p and Wnt5a by dual luciferase reporter gene system.RESULTS AND CONCLUSION:(1)With the prolongation of osteogenic induction time,the level of alkaline phosphatase in cell culture fluid gradually increased.The deposition of dark red or reddish brown calcium salt nodules was gradually increased according to alizarin red staining results.(2)Overexpression of hsa-miR-223-3p decreased the mRNA and protein levels of Wnt5a,OPG and RUNX2 in cells.(3)hsa-miR-223-3p directly targeted the 3'UTR region of Wnt5a and reduced the luciferase activity of Wnt5a.This targeting effect disappeared when the binding site of Wnt5a was mutated.Blocking hsa-miR-223-3p increased the luciferase activity of wild-type Wnt5a,while the luciferase activity of mutant Wnt5a remained unchanged.(4)The results showed that during the differentiation of bone marrow mesenchymal stem cells into osteoblasts,overexpression of hsa-miR-223-3p could affect the expression of Wnt signaling pathway marker Wnt5a and its downstream signaling molecules OPG and RUNX2.hsa-miR-223-3p could directly bind to Wnt5a,which was negatively regulated as a downstream target gene of hsa-miR-223-3p.

关 键 词：干细胞 骨髓间充质干细胞 MIRNA 成骨分化 通路 基因 实验 

分 类 号：R459.9[医药卫生—治疗学] R394.3[医药卫生—临床医学]