Trenkmann Michelle, Brock Matthias, Gay Renate E, Michel Beat A, Gay Steffen, Huber Lars C
Center of Experimental Rheumatology, University Hospital Zurich, Zurich, Switzerland.
Arthritis Rheum. 2013 Apr;65(4):916-27. doi: 10.1002/art.37834.
To elucidate whether the microRNA (miRNA) cluster miR-17-92 contributes to the activated phenotype of rheumatoid arthritis synovial fibroblasts (RASFs).
RASFs were stimulated with tumor necrosis factor α (TNFα), and the expression and regulation of the miR-17-92 cluster were studied using real-time quantitative PCR (PCR) and promoter activity assays. RASFs were transfected with single precursor molecules of miRNAs from miR-17-92 and the expression of matrix-degrading enzymes and cytokines was measured by quantitative PCR and enzyme-linked immunosorbent assay. Potential miRNA targets were identified by computational prediction and were validated using reporter gene assays and Western blotting. The activity of NF-κB signaling was determined by reporter gene assays.
We found that TNFα induces the expression of miR-17-92 in RASFs in an NF-κB-dependent manner. Transfection of RASFs with precursor molecules of single members of miR-17-92 revealed significantly increased expression levels of matrix-degrading enzymes, proinflammatory cytokines, and chemokines in precursor miR-18a (pre-miR-18a)-transfected RASFs. Using reporter gene assays, we identified the NF-κB pathway inhibitor TNFα-induced protein 3 as a new target of miR-18a. In addition, pre-miR-18a-transfected RASFs showed stronger activation of NF-κB signaling, both constitutively and in response to TNFα stimulation.
Our data suggest that the miR-17-92-derived miR-18a contributes to cartilage destruction and chronic inflammation in the joint through a positive feedback loop in NF-κB signaling, with concomitant up-regulation of matrix-degrading enzymes and mediators of inflammation in RASFs.
阐明微小RNA（miRNA）簇miR-17-92是否促成类风湿性关节炎滑膜成纤维细胞（RASFs）的活化表型。
用肿瘤坏死因子α（TNFα）刺激RASFs，采用实时定量聚合酶链反应（PCR）和启动子活性分析研究miR-17-92簇的表达及调控。用来自miR-17-92的单个miRNA前体分子转染RASFs，通过定量PCR和酶联免疫吸附测定法检测基质降解酶和细胞因子的表达。通过计算预测鉴定潜在的miRNA靶标，并使用报告基因测定法和蛋白质印迹法进行验证。通过报告基因测定法确定核因子κB（NF-κB）信号传导的活性。
我们发现TNFα以NF-κB依赖性方式诱导RASFs中miR-17-92的表达。用miR-17-92单个成员的前体分子转染RASFs，发现在前体miR-18a（pre-miR-18a）转染的RASFs中，基质降解酶、促炎细胞因子和趋化因子的表达水平显著增加。使用报告基因测定法，我们确定NF-κB途径抑制剂TNFα诱导蛋白3是miR-18a的新靶标。此外，pre-miR-18a转染的RASFs在组成上以及对TNFα刺激的反应中均显示出更强的NF-κB信号激活。
我们的数据表明，源自miR-17-92的miR-18a通过NF-κB信号传导中的正反馈回路促成关节中的软骨破坏和慢性炎症，同时上调RASFs中基质降解酶和炎症介质的表达。
