miR-15b 通过靶向 Smad7 抑制 BMSCs 成骨分化从而改善 SONFH。
MiR-15b ameliorates SONFH by targeting Smad7 and inhibiting osteogenic differentiation of BMSCs.
机构信息
Department of Orthopedics, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.
出版信息
Eur Rev Med Pharmacol Sci. 2019 Nov;23(22):9761-9771. doi: 10.26355/eurrev_201911_19539.
OBJECTIVE
The aim of this study was to explore the role of micro ribonucleic acid (miR)-15b in steroid-induced osteonecrosis of the femoral head (SONFH) and its potential mechanism.
PATIENTS AND METHODS
Bone marrow tissues were collected from 5 patients with glucocorticoid (GC)-induced ONFH (GC-ONFH, GC group) and 5 patients with secondary ONFH (control group) undergoing total hip replacement in our hospital from July 2016 to August 2017. Subsequently, bone marrow mesenchymal stem cells (BMSCs) were separated from bone marrow extracted and cultured in vitro. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) assay was used to detect differentially expressed miRNAs in BMSCs of patients in GC group and control group. BMSCs were treated with different concentrations of GC. Next, the effect of GC on tmiR-15b expression level was detected via qRT-PCR. Alizarin red staining assay was performed to evaluate the effect of miR-15b on osteogenic differentiation of BMSCs. Meanwhile, the potential targets of miR-15b were predicted using bioinformatics software and validated through luciferase reporter gene assay, respectively. Additionally, Western blotting was conducted to determine the effect of miR-15b on the protein expression of the transforming growth factor beta (TGF-β) signaling pathway.
RESULTS
Flow cytometry demonstrated that the proportion of cluster of differentiation 44 (CD44)-positive cells was 99.7%, while that of CD45-positive cells was only 0.17% in cultured BMSCs. This suggested that the purity of BMSCs was relatively high. QRT-PCR assay indicated that the expression level of miR-15b declined significantly in BMSCs of GC group when compared with control group (p<0.01). The osteogenic differentiation capacity of BMSCs was significantly strengthened in GC group compared with control group (p<0.01). Subsequent qRT-PCR assay revealed that GC down-regulated the expression level of miR-15b in a dose-dependent manner. Besides, the osteogenic differentiation capacity of cells was remarkably strengthened in miR-15b mimic treatment group when compared with control group (p<0.01). Bioinformatics software (TargetScan) predicted that drosophila mothers against decapentaplegic protein 7 (Smad7) might be a potential target of miR-15b, which was indicated by luciferase reporter gene assay. In comparison with control group, miR-15b mimic treatment group exhibited significantly down-regulated protein expression level of Smad7, increased expression level of phosphorylated (p)-Smad2/3 and up-regulated messenger RNA (mRNA) expression level of runt-related transcription factor 2 (Runx2). However, the protein expression level of Smad7 and p-Smad2/3 and the mRNA expression level of Runx2 exhibited opposite trends in miR-15b inhibitor treatment group.
CONCLUSIONS
MiR-15b relieves SONFH by targeting Smad7 and repressing osteogenic differentiation of BMSCs.
目的
本研究旨在探讨微小 RNA-15b（miR-15b）在激素诱导性股骨头坏死（SONFH）中的作用及其潜在机制。
患者与方法
2016 年 7 月至 2017 年 8 月，我院收治的 5 例糖皮质激素（GC）诱导性 ONFH（GC-ONFH，GC 组）和 5 例继发性 ONFH 患者（对照组）接受全髋关节置换术时，采集骨髓组织。随后，从骨髓中分离骨髓间充质干细胞（BMSCs）并在体外培养。采用定量逆转录聚合酶链反应（qRT-PCR）检测 GC 组和对照组患者 BMSCs 中差异表达的 miRNAs。用不同浓度的 GC 处理 BMSCs。然后，通过 qRT-PCR 检测 GC 对 tmiR-15b 表达水平的影响。茜素红染色法评估 miR-15b 对 BMSCs 成骨分化的影响。同时，利用生物信息学软件预测 miR-15b 的潜在靶标，并通过荧光素酶报告基因实验分别进行验证。此外，通过 Western blot 检测 miR-15b 对转化生长因子-β（TGF-β）信号通路蛋白表达的影响。
结果
流式细胞术表明，培养的 BMSCs 中 CD44 阳性细胞的比例为 99.7%，而 CD45 阳性细胞的比例仅为 0.17%。这表明 BMSCs 的纯度相对较高。qRT-PCR 检测结果显示，GC 组 BMSCs 中 miR-15b 的表达水平明显低于对照组（p<0.01）。GC 组 BMSCs 的成骨分化能力明显强于对照组（p<0.01）。随后的 qRT-PCR 检测结果显示，GC 呈剂量依赖性地下调 miR-15b 的表达水平。此外，miR-15b 模拟物处理组细胞的成骨分化能力明显强于对照组（p<0.01）。生物信息学软件（TargetScan）预测 drosophila mothers against decapentaplegic protein 7（Smad7）可能是 miR-15b 的一个潜在靶点，这一预测结果通过荧光素酶报告基因实验得到了证实。与对照组相比，miR-15b 模拟物处理组 Smad7 蛋白表达水平明显下调，磷酸化（p）-Smad2/3 表达水平升高， Runt 相关转录因子 2（Runx2）mRNA 表达水平升高。然而，在 miR-15b 抑制剂处理组中，Smad7 和 p-Smad2/3 的蛋白表达水平以及 Runx2 的 mRNA 表达水平则呈现相反的趋势。
结论
miR-15b 通过靶向 Smad7 抑制 BMSCs 的成骨分化，从而缓解 SONFH。
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