Matsukawa Tetsuya, Sakai Tadahiro, Yonezawa Tomo, Hiraiwa Hideki, Hamada Takashi, Nakashima Motoshige, Ono Yohei, Ishizuka Shinya, Nakahara Hiroyuki, Lotz Martin K, Asahara Hiroshi, Ishiguro Naoki
Arthritis Res Ther. 2013 Feb 13;15(1):R28. doi: 10.1186/ar4164.
Increased expression of aggrecanase-1 (ADAMTS-4) has emerged as an important factor in osteoarthritis (OA) and other joint diseases. This study aimed to determine whether the expression of ADAMTS-4 in human chondrocytes is regulated by miRNA.
MiRNA targets were identified using bioinformatics. Chondrocytes were isolated from knee cartilage and treated with interleukin-1 beta (IL-1β). Gene expression was quantified using TaqMan assays and protein production was determined by immunoblotting. Luciferase reporter assay was used to verify interaction between miRNA and target messenger RNA (mRNA).
In silico analysis predicted putative target sequence of miR-125b on ADAMTS-4. MiR-125b was expressed in both normal and OA chondrocytes, with significantly lower expression in OA chondrocytes than in normal chondrocytes. Furthermore, IL-1β-induced upregulation of ADAMTS-4 was suppressed by overexpression of miR-125b in human OA chondrocytes. In the luciferase reporter assay, mutation of the putative miR-125b binding site in the ADAMTS-4 3'UTR abrogated the suppressive effect of miR125.
Our results indicate that miR-125b plays an important role in regulating the expression of ADAMTS-4 in human chondrocytes and this identifies miR-125b as a novel therapeutic target in OA.
聚集蛋白聚糖酶-1（ADAMTS-4）表达增加已成为骨关节炎（OA）和其他关节疾病的一个重要因素。本研究旨在确定人软骨细胞中ADAMTS-4的表达是否受微小RNA（miRNA）调控。
利用生物信息学鉴定miRNA靶点。从膝关节软骨中分离软骨细胞，并用白细胞介素-1β（IL-1β）处理。使用TaqMan分析法定量基因表达，通过免疫印迹法测定蛋白质产量。荧光素酶报告基因检测用于验证miRNA与靶信使核糖核酸（mRNA）之间的相互作用。
计算机分析预测了miR-125b在ADAMTS-4上的假定靶序列。miR-125b在正常和OA软骨细胞中均有表达，在OA软骨细胞中的表达明显低于正常软骨细胞。此外，在人OA软骨细胞中，miR-125b过表达抑制了IL-1β诱导的ADAMTS-4上调。在荧光素酶报告基因检测中，ADAMTS-4 3'非翻译区（UTR）中假定的miR-125b结合位点的突变消除了miR125的抑制作用。
我们的结果表明，miR-125b在调控人软骨细胞中ADAMTS-4的表达中起重要作用，这表明miR-125b是OA中的一个新的治疗靶点。
