微小RNA-27b调控人骨关节炎软骨细胞中基质金属蛋白酶13的表达。
MicroRNA-27b regulates the expression of matrix metalloproteinase 13 in human osteoarthritis chondrocytes.
作者信息
Akhtar Nahid, Rasheed Zafar, Ramamurthy Sangeetha, Anbazhagan Arivarasu N, Voss Frank R, Haqqi Tariq M
机构信息
School of Medicine, University of South Carolina, Columbia, USA.
出版信息
Arthritis Rheum. 2010 May;62(5):1361-71. doi: 10.1002/art.27329.
OBJECTIVE
Aberrant posttranscriptional regulation of matrix metalloproteinases (MMPs) by microRNA has emerged as an important factor in human diseases. The aim of this study was to determine whether the expression of MMP-13 in human osteoarthritis (OA) chondrocytes is regulated by microRNA.
METHODS
Chondrocytes were stimulated with interleukin-1beta (IL-1beta) in vitro. Total RNA was prepared using TRIzol reagent. Polymerase chain reaction (PCR)-based arrays were used to determine the expression profile of 352 human microRNA. Gene expression was quantified using TaqMan assays, and microRNA targets were identified using bioinformatics. Transfection with reporter construct and microRNA mimic was used to verify suppression of target messenger RNA (mRNA). Gene expression of argonaute and Dicer was determined by reverse transcription-PCR, and expression of protein was determined by immunoblotting. The role of activated MAP kinases (MAPKs) and NF-kappaB was evaluated using specific inhibitors.
RESULTS
In IL-1beta-stimulated OA chondrocytes, 42 microRNA were down-regulated, 2 microRNA were up-regulated, and the expression of 308 microRNA remained unchanged. In silico analysis identified a sequence in the 3'-untranslated region (3'-UTR) of MMP-13 mRNA complementary to the seed sequence of microRNA-27b (miR-27b). Increased expression of MMP-13 correlated with down-regulation of miR-27b. Overexpression of miR-27b suppressed the activity of a reporter construct containing the 3'-UTR of human MMP-13 mRNA and inhibited the IL-1beta-induced expression of MMP-13 protein in chondrocytes. NF-kappaB and MAPK activation down-regulated the expression of miR-27b.
CONCLUSION
Our data demonstrated the expression of miR-27b in both normal and OA chondrocytes. Furthermore, IL-1beta-induced activation of signal transduction pathways associated with the expression of MMP-13 down-regulated the expression of miR-27b. Thus, miR-27b may play a role in regulating the expression of MMP-13 in human chondrocytes.
目的
微小RNA对基质金属蛋白酶（MMPs）的异常转录后调控已成为人类疾病中的一个重要因素。本研究旨在确定微小RNA是否调控人骨关节炎（OA）软骨细胞中MMP - 13的表达。
方法
体外用人白细胞介素 - 1β（IL - 1β）刺激软骨细胞。使用TRIzol试剂制备总RNA。基于聚合酶链反应（PCR）的芯片用于确定352种人类微小RNA的表达谱。使用TaqMan检测法定量基因表达，并使用生物信息学鉴定微小RNA靶标。用报告基因构建体和微小RNA模拟物转染以验证对靶信使核糖核酸（mRNA）的抑制。通过逆转录 - PCR测定AGO蛋白和Dicer的基因表达，并通过免疫印迹测定蛋白表达。使用特异性抑制剂评估活化的丝裂原活化蛋白激酶（MAPK）和核因子κB（NF - κB）的作用。
结果
在IL - 1β刺激的OA软骨细胞中，42种微小RNA下调，2种微小RNA上调，308种微小RNA的表达保持不变。计算机分析在MMP - 13 mRNA的3'非翻译区（3'-UTR）中鉴定出与微小RNA - 27b（miR - 27b）的种子序列互补的序列。MMP - 13表达增加与miR - 27b下调相关。miR - 27b的过表达抑制了含有人类MMP - 13 mRNA 3'-UTR的报告基因构建体的活性，并抑制了软骨细胞中IL - 1β诱导的MMP - 13蛋白表达。NF - κB和MAPK激活下调了miR - 27b的表达。
结论
我们的数据证明了miR - 27b在正常和OA软骨细胞中的表达。此外，IL - 1β诱导的与MMP - 13表达相关的信号转导途径的激活下调了miR - 27b的表达。因此，miR - 27b可能在调节人软骨细胞中MMP - 13的表达中起作用。
Zotero 插件