Role of miR-146a in human chondrocyte apoptosis in response to mechanical pressure injury in vitro.
Role of miR-146a in human chondrocyte apoptosis in response to mechanical pressure injury in vitro.
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DOI：
10.3892/ijmm.2014.1808
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发表时间：
2014-08
影响因子：
5.4
通讯作者：
Ma B
中科院分区：
文献类型：
--
作者：
Jin L;Zhao J;Jing W;Yan S;Wang X;Xiao C;Ma B
已知microRNA（miR）-146a在骨关节炎（OA）中过表达。然而，miR-146 a在OA中的作用尚未完全阐明。在本研究中，我们应用10 MPa的机械压力对人软骨细胞60 min，以研究机械压力损伤后miR-146 a的表达和凋亡。用miR-146 a模拟物或抑制剂转染正常人软骨细胞以调节miR-146 a表达。利用生物信息学方法预测miR-146 a的潜在靶基因。荧光素酶报告基因检测证实Smad 4是miR-146 a的直接靶点。通过定量逆转录PCR和/或蛋白质印迹分析来定量miR-146 a、Smad 4和血管内皮生长因子（VEGF）的表达水平。采用Annexin V-异硫氰酸荧光素（FITC）/碘化丙啶（PI）流式细胞术检测miR-146 a对细胞凋亡的影响。结果表明，机械压力影响软骨细胞的活力，并诱导软骨细胞早期凋亡。机械压力损伤后，软骨细胞中miR-146 a和VEGF的表达水平升高，Smad 4的表达水平降低。在人软骨细胞中，miR-146 a的上调诱导细胞凋亡，上调VEGF表达并下调Smad 4表达。此外，miR-146 a的敲低减少了细胞凋亡，上调了Smad 4表达，下调了VEGF表达。Smad 4通过在其mRNA的3′-非翻译区（3′-UTR）中携带miR-146 a结合序列而被鉴定为miR-146 a的直接靶点。此外，在机械压力损伤的软骨细胞中，miR-146 a诱导的VEGF上调由Smad 4介导。这些结果表明，miR-146 a在我们的实验诱导的人机械损伤的软骨细胞模型中过表达，伴随着VEGF的上调和Smad 4的下调。此外，我们的数据表明，miR-146 a参与了人类软骨细胞对机械损伤的凋亡反应，并可能通过靶向抑制软骨中的Smad 4来增加VEGF水平和破坏转化生长因子（TGF）-β信号通路，从而促进软骨细胞的机械损伤以及OA的发病机制。
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.
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影响因子：
--
作者：
Dossumbekova, Anar;Anghelina, Mirela;Agarwal, Sudha
通讯作者：
Agarwal, Sudha
影响因子：
2.3
作者：
Díaz-Prado S;Cicione C;Muiños-López E;Hermida-Gómez T;Oreiro N;Fernández-López C;Blanco FJ
通讯作者：
Blanco FJ
DOI：
10.1097/00003086-200110001-00009
发表时间：
2001-10-01
影响因子：
4.2
作者：
D'Lima, DD;Hashimoto, S;Lotz, MK
通讯作者：
Lotz, MK
影响因子：
8.8
作者：
通讯作者：
--
Mirbase：MicroRNA基因组学的工具。
影响因子：
14.9
作者：
Griffiths-Jones, Sam;Saini, Harpreet Kaur;van Dongen, Stijn;Enright, Anton J.
通讯作者：
Enright, Anton J.