miR-146a, an IL-1β responsive miRNA, induces vascular endothelial growth factor and chondrocyte apoptosis by targeting Smad4.
miR-146a, an IL-1β responsive miRNA, induces vascular endothelial growth factor and chondrocyte apoptosis by targeting Smad4.
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DOI：
10.1186/ar3798
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发表时间：
2012-04-16
影响因子：
4.9
通讯作者：
Dai K
中科院分区：
文献类型：
--
作者：
Li J;Huang J;Dai L;Yu D;Chen Q;Zhang X;Dai K
miR-146a是最早发现的在骨关节炎（OA）软骨中表达差异的mirna之一。然而，它在OA发病机制中所起的作用尚不清楚。本研究的目的是鉴定miR-146a的分子靶点，从而阐明其在骨性关节炎发病过程中在软骨细胞中的功能。在real-time PCR和/或western blotting检测miR-146a、Smad4和血管内皮生长因子（VEGF）的表达水平之前，用IL-1β处理Sprague-Dawley大鼠的原代软骨细胞。miR-146a对细胞对转化生长因子(TGF -β1)-β1应答的影响通过含有TGF-β1应答元件的荧光素酶报告因子和细胞外信号调节激酶测定来量化。TUNEL法定量检测miR-146a对细胞凋亡的影响。通过手术诱导大鼠关节不稳定的OA发病机制，通过红花素O染色进行组织病理学分析，并通过实时PCR和/或免疫组织化学定量检测miR-146a、Smad4和VEGF的表达水平。IL-1β处理软骨细胞增加了miR-146a和VEGF的表达水平，并以时间依赖性的方式降低了Smad4的水平。miR-146a上调软骨细胞中VEGF的表达，下调Smad4的表达，而miR-146a抑制剂则相反。Smad4是TGF-β途径的常见介质，通过在其mRNA的3'-UTR区域携带miR-146a结合序列，被确定为miR-146a的直接靶标。结合序列的突变显著缓解了miR-146a对Smad4报告基因活性的抑制。此外，miR-146a上调VEGF是由Smad4介导的。miR-146a的表达导致细胞对TGF-β的反应性降低，软骨细胞凋亡率升高。在体内，与假手术组相比，手术诱导的OA大鼠软骨中miR-146a和VEGF的水平更高。相比之下，OA组的Smad4表达水平低于假手术组。IL-1β反应性miR-146a在实验诱导的OA模型中过表达，并伴有VEGF上调和Smad4下调。miR-146a可能通过增加VEGF水平和通过靶向抑制软骨中的Smad4而损害TGF-β信号通路，从而促进OA的发病。
miR-146a is one of the first identified miRNAs expressed differentially in osteoarthritis (OA) cartilage. However, the role it plays in OA pathogenesis is not clear. The aim of this study is to identify a molecular target of miR-146a, thereby elucidating its function in chondrocytes during OA pathogenesis. Primary chondrocytes from Sprague-Dawley rats were treated with IL-1β before the expression levels of miR-146a, Smad4 and vascular endothelial growth factor (VEGF) were quantified by real-time PCR and/or western blotting. The effect of miR-146a on cellular response to transforming growth factor (TGF)-β1 was quantified by a luciferase reporter harboring TGF-β1 responsive elements and by extracellular signal-regulated kinase assay. The effect of miR-146a on apoptosis was quantified by the TUNEL assay. OA pathogenesis was surgically induced with joint instability in rats, evaluated by histopathological analysis with safranin O staining, and the expression levels of miR-146a, Smad4, and VEGF were quantified using real-time PCR and/or immunohistochemistry. IL-1β treatment of chondrocytes increased the expression levels of miR-146a and VEGF and decreased the levels of Smad4 in a time-dependent manner. miR-146a upregulated VEGF expression and downregulated Smad4 expression in chondrocytes, while a miR-146a inhibitor acted in a converse manner. Smad4, a common mediator of the TGF-β pathway, is identified as a direct target of miR-146a by harboring a miR-146a binding sequence in the 3'-UTR region of its mRNA. Mutation of the binding sequence significantly relieved the inhibition of the Smad4 reporter activity by miR-146a. Furthermore, miR-146a upregulation of VEGF is mediated by Smad4. Expression of miR-146a led to a reduction of cellular responsiveness to TGF-β and an increase of apoptosis rate in chondrocytes. In vivo, cartilage from surgically induced OA rats displayed higher levels of miR-146a and VEGF compared with the sham group. In contrast, Smad4 expression level was lower in the OA group than the sham group. IL-1β responsive miR-146a is overexpressed in an experimentally induced OA model, accompanied by upregulation of VEGF and downregulation of Smad4 in vivo. miR-146a may contribute to OA pathogenesis by increasing VEGF levels and by impairing the TGF-β signaling pathway through targeted inhibition of Smad4 in cartilage.
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影响因子：
4.8
作者：
Hayashida, T;deCaestecker, M;Schnaper, HW
通讯作者：
Schnaper, HW
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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.
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作者：
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通讯作者：
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影响因子：
15.9
作者：
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通讯作者：
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