Zhou J, Nie H, Liu P, Wang Z, Yao B, Yang L
Department of Orthopedic, East Campus of Sichuan Provincial People's Hospital, Chengdu, China.
Eur Rev Med Pharmacol Sci. 2019 Jan;23(1):29-36. doi: 10.26355/eurrev_201901_16744.
It was the aim of this study to investigate whether miR-339 may affect osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) by targeting DLX5, thereby alleviating osteoporosis.
BMSCs were isolated from the bone marrow of mice. The expression levels of miR-339 and DLX5 during the process of osteogenesis was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Meanwhile, the expression of downstream osteogenesis-associated proteins, such as runt-related transcription factor 2 (RUNX2) and osteopontin (OPN), were also detected after overexpression or inhibition of miR-339. The alkaline phosphatase (ALP) activity was measured in cells by ALP activity assay kit. Alizarin red staining was performed to reveal the cell mineralization ability. The luciferase reporter gene assay was used to identify the targeted pairings of miR-339 and DLX5 genes. In addition, the expression of DLX5 was detected by Western blot analysis after overexpression or knockdown of miR-339. Rescue test was applied to evaluate whether miR-339 could affect the differentiation of BMSCs by inhibiting the expression of DLX5.
QRT-PCR showed that miR-339 expression gradually decreased while the expression of DLX5 increased during the induction culture of BMSCs. After overexpression of miR-339 in BMSCs, the expression levels of ALP, RUNX2, and OPN were reduced. Besides, ALP activity assay showed a decreased cell ALP activity. RUNX2 protein expression was also decreased. In addition, Alizarin red staining detected a significant increase in cell mineralization, whereas silencing miR-339 resulted in an opposite result. These results indicated that miR-339 could regulate the osteogenic differentiation of BMSCs. Subsequently, we predicted using bioinformatics software that miR-339 might target DLX5, and validated this hypothesis by luciferase reporter assay. Finally, Western blot and ALP activity assay revealed that DLX5 could reverse the inhibitory effect of overexpression of miR-339 on osteogenic differentiation of BMSCs.
Down-regulation of miR-339 can promote osteogenic differentiation of BMSCs by targeting DLX5, thereby relieving osteoporosis.
本研究旨在探讨miR-339是否通过靶向DLX5影响骨髓间充质干细胞（BMSCs）的成骨分化，从而缓解骨质疏松症。
从小鼠骨髓中分离出BMSCs。通过定量实时聚合酶链反应（qRT-PCR）检测成骨过程中miR-339和DLX5的表达水平。同时，在过表达或抑制miR-339后，检测下游成骨相关蛋白如 runt相关转录因子2（RUNX2）和骨桥蛋白（OPN）的表达。用碱性磷酸酶（ALP）活性检测试剂盒测定细胞中的ALP活性。进行茜素红染色以揭示细胞矿化能力。用荧光素酶报告基因检测法鉴定miR-339与DLX5基因的靶向配对。此外，在过表达或敲低miR-339后，通过蛋白质免疫印迹分析检测DLX5的表达。应用拯救试验评估miR-339是否可通过抑制DLX5的表达影响BMSCs的分化。
qRT-PCR显示，在BMSCs诱导培养过程中，miR-339表达逐渐降低，而DLX5表达增加。在BMSCs中过表达miR-339后，ALP、RUNX2和OPN的表达水平降低。此外，ALP活性检测显示细胞ALP活性降低。RUNX2蛋白表达也降低。另外，茜素红染色检测到细胞矿化显著增加，而沉默miR-339则产生相反结果。这些结果表明miR-339可调节BMSCs的成骨分化。随后，我们使用生物信息学软件预测miR-339可能靶向DLX5，并通过荧光素酶报告基因检测验证了这一假设。最后，蛋白质免疫印迹和ALP活性检测显示，DLX5可逆转miR-339过表达对BMSCs成骨分化的抑制作用。
miR-339的下调可通过靶向DLX5促进BMSCs的成骨分化，从而缓解骨质疏松症。
