Department of Orthopedics, The Affiliated Hospital (Traditional Chinese Medicine) of Southwest Medical University, Luzhou, China.
Eur Rev Med Pharmacol Sci. 2019 Aug;23(16):6791-6799. doi: 10.26355/eurrev_201908_18717.
To explore whether microRNA-579-3P was involved in the development of osteoporosis, and to investigate the possible molecular mechanisms.
The messenger RNA (mRNA) expression levels of microRNA-579-3P, alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2) and bone sialoprotein (BSP) in serum samples of osteoporosis patients and normal controls were detected by quantitative Real-time polymerase chain reaction (qRT-PCR), respectively. Meanwhile, the expressions of the above genes during osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs) were examined as well. To investigate the effect of microRNA-579-3P on osteogenesis, microRNA-579-3P was overexpressed and knocked down in hMSCs. Subsequently, the mRNA and protein expression levels of osteogenesis-related genes, such as ALP, RUNX2 and BSP, were detected by qRT-PCR and Western blot, respectively. In addition, ALP activity and mineralization forming ability were evaluated by ALP staining and alizarin red staining. Bioinformatics predicted that Sirt1 was the target gene of microRNA-579-3P. Subsequent luciferase reporter gene assay was performed to verify the binding relationship of microRNA-579-3P to Sirt1. Meanwhile, qRT-PCR and Western blot were used to detect the changes in the mRNA and protein expression levels of Sirt1, respectively. After overexpression of microRNA-579-3P and Sirt1, qRT-PCR, Western blot, ALP staining and alizarin red staining assays were performed to detect the osteogenic differentiation of hMSCs.
The expression of microRNA-579-3P in serum of patients with osteoporosis was significantly higher than that of normal controls. Meanwhile, the expression of microRNA-579-3P decreased gradually during osteogenic differentiation of hMSCs. Overexpression of microRNA-579-3P significantly reduced the expressions of osteogenic related genes, including ALP, RUNX2 and BSP. Besides, ALP activity and mineralized nodule formation ability decreased obviously as well. Luciferase reporter gene assay showed that microRNA-579-3P could bind to Sirt1. After overexpression of microRNA-579-3P, the mRNA and protein expression levels of Sirt1 were significantly reduced, which were reversed after silence of microRNA-579-3P. Simultaneous overexpression of microRNA-579-3P and Sirt1 could reverse the inhibition of osteogenic differentiation of hMSCs caused by overexpression of microRNA-579-3P alone.
MicroRNA-579-3P could inhibit osteogenic differentiation of hMSCs by regulating Sirt1, thereby promoting the development of osteoporosis.
探讨 microRNA-579-3P 是否参与骨质疏松症的发生发展，并探讨其可能的分子机制。
采用实时定量聚合酶链反应(qRT-PCR)分别检测骨质疏松症患者和正常对照者血清中 microRNA-579-3P、碱性磷酸酶(ALP)、 runt 相关转录因子 2(RUNX2)和骨涎蛋白(BSP)的信使 RNA(mRNA)表达水平。同时，检测人骨髓间充质干细胞(hMSCs)成骨分化过程中上述基因的表达情况。为了研究 microRNA-579-3P 对成骨的影响，在 hMSCs 中转染 microRNA-579-3P 过表达和敲低载体。随后，采用 qRT-PCR 和 Western blot 分别检测成骨相关基因(如 ALP、RUNX2 和 BSP)的 mRNA 和蛋白表达水平。此外，通过碱性磷酸酶染色和茜素红染色评估 ALP 活性和矿化形成能力。生物信息学预测 Sirt1 是 microRNA-579-3P 的靶基因。随后进行荧光素酶报告基因实验验证 microRNA-579-3P 与 Sirt1 的结合关系。同时，采用 qRT-PCR 和 Western blot 分别检测 Sirt1 的 mRNA 和蛋白表达水平的变化。过表达 microRNA-579-3P 和 Sirt1 后，进行 qRT-PCR、Western blot、碱性磷酸酶染色和茜素红染色实验，检测 hMSCs 的成骨分化情况。
骨质疏松症患者血清中 microRNA-579-3P 的表达明显高于正常对照组。同时，在 hMSCs 成骨分化过程中，microRNA-579-3P 的表达逐渐降低。microRNA-579-3P 过表达显著降低了成骨相关基因(包括 ALP、RUNX2 和 BSP)的表达。此外，ALP 活性和矿化结节形成能力明显降低。荧光素酶报告基因实验表明，microRNA-579-3P 可与 Sirt1 结合。过表达 microRNA-579-3P 后，Sirt1 的 mRNA 和蛋白表达水平明显降低，沉默 microRNA-579-3P 后则逆转。同时过表达 microRNA-579-3P 和 Sirt1 可逆转 microRNA-579-3P 过表达单独引起的 hMSCs 成骨分化抑制。
microRNA-579-3P 可能通过调节 Sirt1 抑制 hMSCs 的成骨分化，从而促进骨质疏松症的发生发展。
