【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。更多还原

【Abstract】 BACKGROUND: Studies have found that miR-223-3 p is highly expressed in osteoporosis patients, but the related mechanism is rarely studied.OBJECTIVE: To investigate the regulatory effect of hsa-miR-223-3 p 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-223-3 p. RT-qPCR and western blot assay were used to detect the changes in the mRNA and protein levels of Wnt signaling pathway markers Wnt5 a and its downstream signaling molecules OPG and RUNX2. PmirGLO/Wnt5 a-3’UTR and pmirGLO/Wnt5 a-3’UTR mut plasmids were constructed and co-transfected with hsa-miR-223-3 p mimics/control plasmid into 293 T cells to verify the targeting relationship between hsa-miR-223-3 p and Wnt5 a 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-223-3 p decreased the mRNA and protein levels of Wnt5 a, OPG and RUNX2 in cells.(3) hsa-miR-223-3 p directly targeted the 3’UTR region of Wnt5 a and reduced the luciferase activity of Wnt5 a. This targeting effect disappeared when the binding site of Wnt5 a was mutated. Blocking hsa-miR-223-3 p increased the luciferase activity of wild-type Wnt5 a, while the luciferase activity of mutant Wnt5 a remained unchanged.(4) The results showed that during the differentiation of bone marrow mesenchymal stem cells into osteoblasts, overexpression of hsa-miR-223-3 p could affect the expression of Wnt signaling pathway marker Wnt5 a and its downstream signaling molecules OPG and RUNX2. hsa-miR-223-3 p could directly bind to Wnt5 a, which was negatively regulated as a downstream target gene of hsa-miR-223-3 p.更多还原