MiR-20a regulates ASK1 expression and TLR4-dependent cytokine release in rheumatoid fibroblast-like synoviocytes
MiR-20a regulates ASK1 expression and TLR4-dependent cytokine release in rheumatoid fibroblast-like synoviocytes
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DOI：
10.1136/annrheumdis-2012-201654
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发表时间：
2013-06-01
影响因子：
27.4
通讯作者：
Georgel, Philippe
中科院分区：
文献类型：
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作者：
Philippe, Lucas;Alsaleh, Ghada;Georgel, Philippe
目的探讨类风湿性关节炎（RA）成纤维细胞样滑膜细胞（FLS）中miR-17-92簇中的miR-20 a是否通过调节toll样受体4通路中凋亡信号调节激酶（ASK）1的表达，方法采用RT-qPCR方法检测miR-20 a和ASK 1 mRNA在p38丝裂原活化蛋白激酶上游的表达。Western blotting检测ASK 1蛋白表达。通过用miR-20 a模拟物转染RA FLS和THP-1细胞来进行miR-20 a的过表达。通过ELISA测量白细胞介素（IL）-6、CXCL-10、IL-1 β和TNF-α的释放。miR-20 a在体内的作用进行了评估，从腹腔注射的小鼠获得的巨噬细胞的IL-6释放与vectorised miR-20 a mimics.Results我们表明，RA FLS与脂多糖（LPS）和细菌脂蛋白（BLP）的刺激诱导miR-20 a的表达下降，这种下降与ASK 1的表达上调。使用Ask 1 3 'UTR报告基因的转染，我们证明了Ask 1是miR-20 a的直接靶点。miR-20 a的过表达导致BLP和LPS激活的细胞中ASK 1蛋白的整体减少，表明miR-20 a在翻译水平上调节ASK 1的表达。miR-20 a模拟物的转染降低了RA FLS释放IL-6和CXCL 10以及活化的THP-1细胞释放IL-1 β和TNF-α，但仅响应于LPS。最后，注射载体化的miR-20 a模拟物的小鼠导致了一个整体的降低，在ASK 1蛋白表达和IL-6分泌LPS激活的macrophages.Conclusions我们的数据点对一个重要的作用，miR-20 a的调节促炎细胞因子的释放，通过控制ASK 1在RA FLS的表达。
Objective To evaluate whether miR-20a belonging to the cluster miR-17-92 is a negative regulator of inflammation in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) by modulating expression of apoptosis signal-regulating kinase (ASK) 1, a key component of the toll-like receptors 4 pathway, upstream of p38 mitogen-activated protein kinase.Methods Evaluation of miR-20a and ASK1 mRNA was performed by RT-qPCR. ASK1 protein expression was assessed by western blotting. Overexpression of miR-20a was performed by transfection of RA FLS and THP-1 cells with miR-20a mimics. Interleukin (IL)-6, CXCL-10, IL-1 beta and TNF-alpha release were measured by ELISA. The role of miR-20a in vivo was assessed by IL-6 release from macrophages obtained from mice injected intraperitoneally with vectorised miR-20a mimics.Results We showed that stimulation of RA FLS with lipopolysacharide (LPS) and bacterial lipoproteins (BLP) induces a drop in expression of miR-20a and that this decrease is associated with an upregulation of ASK1 expression. Using transfection of Ask1 3'UTR reporters, we demonstrate that Ask1 is a direct target of miR-20a. Overexpression of miR-20a led to a global decrease in ASK1 protein in BLP- and LPS-activated cells indicating that miR-20a regulates the expression of ASK1 at the translational level. Transfection of miR-20a mimics decreases IL-6 and CXCL10 release by RA FLS and IL-1 beta and TNF-alpha by activated THP-1 cells but only in response to LPS. Last, injection of vectorised miR-20a mimics to mice led to a global decrease in ASK1 protein expression and IL-6 secretion in LPS-activated macrophages.Conclusions Our data point toward an important role for miR-20a in the regulation of pro-inflammatory cytokines release, by controlling ASK1 expression in RA FLS.