Department of Traumatology, Yantaishan Hospital, Yantai, China.
Eur Rev Med Pharmacol Sci. 2019 Jan;23(2):456-463. doi: 10.26355/eurrev_201901_16855.
To explore the role of microRNA-9-5p in regulating osteoporosis (OS) development and its underlying mechanism.
MicroRNA-9-5p expression in peripheral blood of 30 OS patients and 30 healthy subjects was examined by quantitative Real-Time-Polymerase Chain Reaction (qRT-PCR). During the processes of osteogenesis and adipogenesis, mRNA levels of microRNA-9-5p, osteogenesis-related genes, and adipogenesis-related genes in marrow stromal stem cells (MSCs) were detected by qRT-PCR as well. After overexpression or knockdown of microRNA-9-5p, the regulatory effects of microRNA-9-5p on osteogenesis-related genes and adipogenesis-related genes in MSCs were accessed by detecting their mRNA and protein levels. Alizarin red staining and oil red staining were performed to determine the osteogenic and adipogenic capacities of MSCs after microRNA-9-5p overexpression, respectively. The dual-luciferase reporter gene assay was conducted to verify the binding condition of microRNA-9-5p and Wnt3a. Finally, rescue experiments were performed to confirm whether microRNA-9-5p could regulate OS development via targeting Wnt3a.
Higher expression of microRNA-9-5p was found in OS patients than that of healthy controls. MicroRNA-9-5p expression was downregulated with the prolongation of osteogenic induction, whereas it was upregulated during the process of adipogenic differentiation. Overexpression of microRNA-9-5p downregulated mRNA levels of osteogenesis-related genes (ALP, RUNX2, and OPN), whereas upregulated adipogenesis-related genes (PPARγ, Adipsin, and C/EBPα) in MSCs. The number of calcified nodules became fewer after microRNA-9-5p overexpression in MSCs. MSCs that overexpressed microRNA-9-5p showed more lipid droplets than that of controls. Subsequently, the dual-luciferase reporter gene assay verified that Wnt3a is the target gene of microRNA-9-5p. Both mRNA and protein levels of Wnt3a were negatively regulated by microRNA-9-5p. Rescue experiments indicated that the regulatory effects of microRNA-9-5p on osteogenesis and adipogenesis of MSCs were reversed by Wnt3a overexpression.
MicroRNA-9-5p is lowly expressed in the peripheral blood of OS patients. MicroRNA-9-5p promotes the occurrence and progression of OS through inhibiting osteogenesis and promoting adipogenesis via targeting Wnt3a.
探讨 microRNA-9-5p 在调控骨质疏松症（OS）发生发展中的作用及其机制。
采用实时荧光定量聚合酶链反应（qRT-PCR）检测 30 例 OS 患者和 30 例健康对照者外周血中 microRNA-9-5p 的表达，同时检测骨髓基质干细胞（MSCs）成骨和成脂分化过程中 microRNA-9-5p 及成骨相关基因、成脂相关基因的 mRNA 水平。过表达或敲低 microRNA-9-5p 后，检测 MSCs 中 microRNA-9-5p 对成骨相关基因和成脂相关基因的调控作用，通过检测其 mRNA 和蛋白水平来实现。茜素红染色和油红染色分别用于确定 MSCs 经 microRNA-9-5p 过表达后的成骨和成脂能力。双荧光素酶报告基因检测用于验证 microRNA-9-5p 与 Wnt3a 的结合情况。最后，通过进行 rescue 实验来确认 microRNA-9-5p 是否可以通过靶向 Wnt3a 来调节 OS 的发生发展。
OS 患者外周血中 microRNA-9-5p 表达水平高于健康对照组。随着成骨诱导时间的延长，microRNA-9-5p 的表达下调，而在成脂分化过程中则上调。过表达 microRNA-9-5p 可下调 MSCs 中成骨相关基因（ALP、RUNX2 和 OPN）的 mRNA 水平，而上调成脂相关基因（PPARγ、Adipsin 和 C/EBPα）。MSCs 中过表达 microRNA-9-5p 后，钙化结节数量减少。过表达 microRNA-9-5p 的 MSCs 比对照组的脂滴更多。随后，双荧光素酶报告基因检测证实 Wnt3a 是 microRNA-9-5p 的靶基因。microRNA-9-5p 负调控 Wnt3a 的 mRNA 和蛋白水平。Rescue 实验表明，Wnt3a 过表达可逆转 microRNA-9-5p 对 MSCs 成骨和成脂的调节作用。
OS 患者外周血中 microRNA-9-5p 表达水平较低。microRNA-9-5p 通过靶向 Wnt3a 抑制成骨、促进成脂，从而促进 OS 的发生发展。
