微小RNA-29a通过靶向转化生长因子-β激活激酶1结合蛋白1降低真皮成纤维细胞中金属蛋白酶组织抑制因子-1的产生，对系统性硬化症具有重要意义。
MiR-29a reduces TIMP-1 production by dermal fibroblasts via targeting TGF-β activated kinase 1 binding protein 1, implications for systemic sclerosis.
作者信息
Ciechomska Marzena, O'Reilly Steven, Suwara Monika, Bogunia-Kubik Katarzyna, van Laar Jacob M
机构信息
Newcastle University, Musculoskeletal Research Group, Institute of Cellular Medicine, Newcastle upon Tyne, United Kingdom; L. Hirszferd Institute of Immunology and Experimental Therapy, Polish Academy of Science, Wroclaw, Poland.
Newcastle University, Musculoskeletal Research Group, Institute of Cellular Medicine, Newcastle upon Tyne, United Kingdom.
出版信息
PLoS One. 2014 Dec 30;9(12):e115596. doi: 10.1371/journal.pone.0115596. eCollection 2014.
BACKGROUND
Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterised by skin and internal organs fibrosis due to accumulation of extra cellular matrix (ECM) proteins. Tissue inhibitor of metalloproteinases 1 (TIMP-1) plays a key role in ECM deposition.
AIM
To investigate the role of miR-29a in regulation of TAB1-mediated TIMP-1 production in dermal fibroblasts in systemic sclerosis.
METHODS
Healthy control (HC) and SSc fibroblasts were cultured from skin biopsies. The expression of TIMP-1, MMP-1 and TGF-β activated kinase 1 binding protein 1 (TAB1) was measured following miR-29a transfection using ELISA, qRT-PCR, and Western Blotting. The functional effect of miR-29a on dermal fibroblasts was assessed in collagen gel assay. In addition, HeLa cells were transfected with 3'UTR of TAB1 plasmid cloned downstream of firefly luciferase gene to assess TAB1 activity. HC fibroblasts and HeLa cells were also transfected with Target protectors in order to block the endogenous miR-29a activity.
RESULTS
We found that TAB1 is a novel target gene of miR-29a, also regulating downstream TIMP-1 production. TAB1 is involved in TGF-β signal transduction, a key cytokine triggering TIMP-1 production. To confirm that TAB1 is a bona fide target gene of miR-29a, we used a TAB1 3'UTR luciferase assay and Target protector system. We showed that miR-29a not only reduced TIMP-1 secretion via TAB1 repression, but also increased functional MMP-1 production resulting in collagen degradation. Blocking TAB1 activity by pharmacological inhibition or TAB1 knockdown resulted in TIMP-1 reduction, confirming TAB1-dependent TIMP-1 regulation. Enhanced expression of miR-29a was able to reverse the profibrotic phenotype of SSc fibroblasts via downregulation of collagen and TIMP-1.
CONCLUSIONS
miR-29a repressed TAB1-mediated TIMP-1 production in dermal fibroblasts, demonstrating that miR-29a may be a therapeutic target in SSc.
背景
系统性硬化症（SSc）是一种自身免疫性结缔组织疾病，其特征是由于细胞外基质（ECM）蛋白积累导致皮肤和内脏器官纤维化。金属蛋白酶组织抑制剂1（TIMP-1）在ECM沉积中起关键作用。
目的
研究miR-29a在系统性硬化症中对真皮成纤维细胞中TAB1介导的TIMP-1产生的调节作用。
方法
从皮肤活检组织中培养健康对照（HC）和SSc成纤维细胞。使用ELISA、qRT-PCR和蛋白质免疫印迹法检测miR-29a转染后TIMP-1、MMP-1和转化生长因子-β激活激酶1结合蛋白1（TAB1）的表达。通过胶原凝胶试验评估miR-29a对真皮成纤维细胞的功能影响。此外，将TAB1质粒的3'UTR克隆到萤火虫荧光素酶基因下游转染HeLa细胞，以评估TAB1活性。HC成纤维细胞和HeLa细胞也用靶向保护剂转染，以阻断内源性miR-29a活性。
结果
我们发现TAB1是miR-29a的一个新靶基因，也调节下游TIMP-1的产生。TAB1参与转化生长因子-β信号转导，转化生长因子-β是触发TIMP-1产生的关键细胞因子。为了证实TAB1是miR-29a的真正靶基因，我们使用了TAB1 3'UTR荧光素酶试验和靶向保护剂系统。我们表明，miR-29a不仅通过抑制TAB1减少TIMP-1分泌，还增加功能性MMP-1的产生，导致胶原蛋白降解。通过药物抑制或敲低TAB1活性导致TIMP-1减少，证实了TAB1依赖性TIMP-1调节。miR-29a的表达增强能够通过下调胶原蛋白和TIMP-1来逆转SSc成纤维细胞的促纤维化表型。
结论
miR-29a抑制真皮成纤维细胞中TAB1介导的TIMP-1产生，表明miR-29a可能是系统性硬化症的一个治疗靶点。
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