MicroRNA-27b regulates the expression of matrix metalloproteinase 13 in human osteoarthritis chondrocytes.
MicroRNA-27b regulates the expression of matrix metalloproteinase 13 in human osteoarthritis chondrocytes.
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DOI：
10.1002/art.27329
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发表时间：
2010-05
影响因子：
--
通讯作者：
Haqqi, Tariq M.
中科院分区：
文献类型：
--
作者：
Akhtar, Nahid;Rasheed, Zafar;Ramamurthy, Sangeetha;Anbazhagan, Arivarasu N.;Voss, Frank R.;Haqqi, Tariq M.
microRNA对基质金属蛋白酶（MMPs）的转录后异常调控已成为人类疾病的重要因素。本研究的目的是确定人类骨关节炎（OA）软骨细胞中MMP-13的表达是否受microRNA的调控。用白细胞介素-1 β（IL-1β）体外刺激软骨细胞。使用TRIzol试剂制备总RNA。使用基于聚合酶链反应（PCR）的阵列来确定352种人类microRNA的表达谱。使用TaqMan测定法定量基因表达，并使用生物信息学鉴定microRNA靶标。使用报告构建体和微小RNA模拟物转染来验证靶信使RNA（mRNA）的抑制。通过逆转录-PCR测定argonaute和Dicer的基因表达，并通过免疫印迹测定蛋白质的表达。使用特异性抑制剂评估活化的MAP激酶（MAPKs）和NF-κB的作用。在IL-1β刺激的OA软骨细胞中，42种microRNA表达下调，2种microRNA表达上调，308种microRNA表达不变。计算机模拟分析在MMP-13 mRNA的3′-非翻译区（3′-UTR）中鉴定出与microRNA-27 b（miR-27 b）的种子序列互补的序列。MMP-13的表达增加与miR-27 b的下调相关。过表达miR-27 b可抑制含有人MMP-13 mRNA 3′-UTR的报告基因的活性，并抑制IL-1β诱导的软骨细胞MMP-13蛋白的表达。NF-κB和MAPK激活下调miR-27 B的表达。我们的数据表明miR-27 b在正常和OA软骨细胞中均有表达。此外，IL-1β激活了与MMP-13表达相关的信号转导通路，下调了miR-27 b的表达。因此，miR-27 b可能在调节人软骨细胞中MMP-13的表达中起作用。
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. Chondrocytes were stimulated with interleukin-1β (IL-1β) 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-κB was evaluated using specific inhibitors. In IL-1β–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-1β–induced expression of MMP-13 protein in chondrocytes. NF-κB and MAPK activation down-regulated the expression of miR-27b. Our data demonstrated the expression of miR-27b in both normal and OA chondrocytes. Furthermore, IL-1β–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.
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