Department of Orthopedics, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710038, P.R. China.
Int J Mol Med. 2014 Aug;34(2):451-63. doi: 10.3892/ijmm.2014.1808. Epub 2014 Jun 16.
MicroRNA (miR)-146a is known to be overexpressed in osteoarthritis (OA). However, the role of miR-146a in OA has not yet been fully elucidated. In the present study, we applied mechanical pressure of 10 MPa to human chondrocytes for 60 min in order to investigate the expression of miR-146a and apoptosis following the mechanical pressure injury. Normal human chondrocytes were transfected with an miR-146a mimic or an inhibitor to regulate miR-146a expression. Potential target genes of miR-146a were predicted using bioinformatics. Moreover, luciferase reporter assay confirmed that Smad4 was a direct target of miR-146a. The expression levels of miR-146a, Smad4 and vascular endothelial growth factor (VEGF) were quantified by quantitative reverse transcription PCR and/or western blot analysis. The effects of miR-146a on apoptosis were detected by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry. The results indicated that mechanical pressure affected chondrocyte viability and induced the early apoptosis of chondrocytes. Mechanical pressure injury increased the expression levels of miR-146a and VEGF and decreased the levels of Smad4 in the chondrocytes. In the human chondrocytes, the upregulation of miR-146a induced apoptosis, upregulated VEGF expression and downregulated Smad4 expression. In addition, the knockdown of miR-146a reduced cell apoptosis, upregulated Smad4 expression and downregulated VEGF expression. Smad4 was identified as a direct target of miR-146a by harboring a miR‑146a binding sequence in the 3'-untranslated region (3'-UTR) of its mRNA. Furthermore, the upregulation of VEGF induced by miR‑146a was mediated by Smad4 in the chondrocytes subjected to mechanical pressure injury. These results demonstrated that miR-146a was overexpressed in our chondrocyte model of experimentally induced human mechanical injury, accompanied by the upregulation of VEGF and the downregulation of Smad4 in vitro. Moreover, our data suggest that miR-146a is involved in human chondrocyte apoptosis in response to mechanical injury, and may contribute to the mechanical injury of chondrocytes, as well as to the pathogenesis of OA by increasing the levels of VEGF and damaging the transforming growth factor (TGF)-β signaling pathway through the targeted inhibition of Smad4 in cartilage.
微小 RNA（miR）-146a 在骨关节炎（OA）中被证实过度表达。然而，miR-146a 在 OA 中的作用尚未完全阐明。在本研究中，我们对人软骨细胞施加 10 MPa 的机械压力 60 分钟，以研究机械压力损伤后 miR-146a 的表达和细胞凋亡情况。用 miR-146a 模拟物或抑制剂转染正常人类软骨细胞以调节 miR-146a 的表达。使用生物信息学预测 miR-146a 的潜在靶基因。此外，荧光素酶报告基因实验证实 Smad4 是 miR-146a 的直接靶基因。通过定量逆转录 PCR 和/或 Western blot 分析来定量 miR-146a、Smad4 和血管内皮生长因子（VEGF）的表达水平。通过 Annexin V-荧光素异硫氰酸酯（FITC）/碘化丙啶（PI）流式细胞术检测 miR-146a 对细胞凋亡的影响。结果表明，机械压力影响软骨细胞活力并诱导软骨细胞早期凋亡。机械压力损伤增加了 miR-146a 和 VEGF 的表达水平，并降低了软骨细胞中 Smad4 的水平。在人软骨细胞中，上调 miR-146a 诱导细胞凋亡，上调 VEGF 表达，下调 Smad4 表达。此外，下调 miR-146a 减少细胞凋亡，上调 Smad4 表达，下调 VEGF 表达。Smad4 在其 mRNA 的 3'-非翻译区（3'-UTR）中具有 miR-146a 结合序列，被鉴定为 miR-146a 的直接靶基因。此外，在机械压力损伤的软骨细胞中，miR-146a 诱导的 VEGF 上调是通过 Smad4 介导的。这些结果表明，miR-146a 在我们的体外人机械损伤诱导的软骨细胞模型中过度表达，伴随着 VEGF 的上调和 Smad4 的下调。此外，我们的数据表明，miR-146a 参与了人软骨细胞对机械损伤的凋亡反应，并且可能通过靶向抑制软骨中的 Smad4 来增加 VEGF 的水平并破坏转化生长因子（TGF）-β 信号通路，从而导致软骨细胞的机械损伤，并导致 OA 的发病机制。
