Maurer Britta, Stanczyk Joanna, Jüngel Astrid, Akhmetshina Alfiya, Trenkmann Michelle, Brock Matthias, Kowal-Bielecka Otylia, Gay Renate E, Michel Beat A, Distler Jörg H W, Gay Steffen, Distler Oliver
University Hospital Zurich and Zurich Center of Integrative Human Physiology, Zurich, Switzerland.
Arthritis Rheum. 2010 Jun;62(6):1733-43. doi: 10.1002/art.27443.
To investigate the role of microRNA (miRNA) as posttranscriptional regulators of profibrotic genes in systemic sclerosis (SSc).
MicroRNA, which target collagens, were identified by in silico analysis. Expression of miRNA-29 (miR-29) was determined by TaqMan real-time polymerase chain reaction analysis of skin biopsy and fibroblast samples from SSc patients and healthy controls as well as in the mouse model of bleomycin-induced skin fibrosis. Cells were transfected with precursor miRNA (pre-miRNA)/anti-miRNA of miR-29 using Lipofectamine. Collagen gene expression was also studied in luciferase reporter gene assays. For stimulation, recombinant transforming growth factor beta (TGFbeta), platelet-derived growth factor B (PDGF-B), or interleukin-4 (IL-4) was used. The effects of inhibiting PDGF-B and TGFbeta signaling on the levels of miR-29 were studied in vitro and in the bleomycin model.
We found that miR-29a was strongly down-regulated in SSc fibroblasts and skin sections as compared with the healthy controls. Overexpression in SSc fibroblasts significantly decreased, and accordingly, knockdown in normal fibroblasts increased, the levels of messenger RNA and protein for type I and type III collagen. In the reporter gene assay, cotransfection with pre-miR-29a significantly decreased the relative luciferase activity, which suggests a direct regulation of collagen by miR-29a. TGFbeta, PDGF-B, or IL-4 reduced the levels of miR-29a in normal fibroblasts to those seen in SSc fibroblasts. Similar to human SSc, the expression of miR-29a was reduced in the bleomycin model of skin fibrosis. Inhibition of PDGF-B and TGFbeta pathways by treatment with imatinib restored the levels of miR-29a in vitro and in the bleomycin model in vivo.
These data add the posttranscriptional regulation of collagens by miR-29a as a novel aspect to the fibrogenesis of SSc and suggest miR-29a as a potential therapeutic target.
研究微小RNA（miRNA）作为系统性硬化症（SSc）中促纤维化基因转录后调节因子的作用。
通过计算机分析鉴定靶向胶原蛋白的微小RNA。采用TaqMan实时聚合酶链反应分析，测定SSc患者和健康对照者的皮肤活检及成纤维细胞样本以及博来霉素诱导的皮肤纤维化小鼠模型中miRNA-29（miR-29）的表达。使用Lipofectamine将miR-29的前体微小RNA（pre-miRNA）/抗微小RNA转染至细胞。还通过荧光素酶报告基因测定研究胶原蛋白基因表达。为进行刺激，使用重组转化生长因子β（TGFβ）、血小板衍生生长因子B（PDGF-B）或白细胞介素-4（IL-4）。在体外和博来霉素模型中研究抑制PDGF-B和TGFβ信号传导对miR-29水平的影响。
我们发现与健康对照相比，miR-29a在SSc成纤维细胞和皮肤切片中强烈下调。在SSc成纤维细胞中过表达显著降低，相应地，在正常成纤维细胞中敲低则增加I型和III型胶原蛋白的信使核糖核酸和蛋白质水平。在报告基因测定中，与pre-miR-29a共转染显著降低相对荧光素酶活性，这表明miR-29a对胶原蛋白有直接调节作用。TGFβ、PDGF-B或IL-4将正常成纤维细胞中miR-29a的水平降低至SSc成纤维细胞中的水平。与人类SSc相似，在皮肤纤维化的博来霉素模型中miR-29a的表达降低。用伊马替尼治疗抑制PDGF-B和TGFβ途径可在体外和体内博来霉素模型中恢复miR-29a的水平。
这些数据为miR-29a对胶原蛋白的转录后调节作为SSc纤维化形成的一个新方面提供了依据，并提示miR-29a作为一个潜在的治疗靶点。
