Mao Guping, Wu Peihui, Zhang Ziji, Zhang Zhiqi, Liao Weiming, Li Yukang, Kang Yan
Department of Joint Surgery, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.
Tower Hill School, Wilmington, Delaware, USA.
Cell Physiol Biochem. 2017;44(1):38-52. doi: 10.1159/000484579. Epub 2017 Nov 3.
BACKGROUND/AIMS: Aggrecanase-1 (ADAMTS-4) and aggrecanase-2 (ADAMTS-5) are secreted enzymes belonging to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family that play significant roles in the progression of osteoarthritis (OA). Here, we aimed to determine whether the expression of ADAMTS-4/5 in chondrogenesis and inflammation is regulated by microRNA-92a-3p (miR-92a-3p).
MiR-92a-3p and ADAMTS-4/5 expressions were determined by quantitative polymerase chain reaction (qPCR). To investigate the repressive effect of miR-92a-3p on ADAMTS-4/5 expression, chondrogenic human mesenchymal stem cells (hMSCs) and human chondrocytes were transfected with mature miR-92a-3p or an antisense inhibitor (anti-miR-92a-3p), respectively. ADAMTS-4/5 protein production was quantified by enzyme-linked immunosorbent assay (ELISA), and miR-92a-3p involvement in IL-1β-mediated catabolic effects was examined by immunoblotting. The roles of activated MAP kinases (MAPK) and nuclear factor (NF)-κB were evaluated by using specific inhibitors. Interaction between miR-92a-3p and its putative binding site in the 3'-untranslated region (3'-UTR) of ADAMTS-4/5 mRNA was confirmed by luciferase reporter assay.
miR-92a-3p expression was elevated in chondrogenic hMSCs, with significantly lower expression in OA cartilage than in normal cartilage. Stimulation with IL-1β significantly reduced miR-92a-3p expression in primary human chondrocytes (PHCs). Transfection of chondrocytes with miR-92a-3p downregulated IL-1β-induced ADAMTS-4/5 expression, and the activity of a reporter construct containing the 3'-UTR of human ADAMTS-4/5 mRNA. MiR-92a-3p expression was suppressed upon IL-1β-induced activation of MAPK and NF-κB in chondrocytes.
MiR-92a-3p is an important regulator of ADAMTS-4/5 in human chondrocytes and may contribute to the development of OA.
背景/目的：聚集蛋白聚糖酶-1（ADAMTS-4）和聚集蛋白聚糖酶-2（ADAMTS-5）是属于ADAMTS（含血小板反应蛋白基序的解聚素和金属蛋白酶）家族的分泌酶，在骨关节炎（OA）进展中起重要作用。在此，我们旨在确定微小RNA-92a-3p（miR-92a-3p）是否调控软骨形成和炎症过程中ADAMTS-4/5的表达。
通过定量聚合酶链反应（qPCR）测定miR-92a-3p和ADAMTS-4/5的表达。为研究miR-92a-3p对ADAMTS-4/5表达的抑制作用，分别用成熟miR-92a-3p或反义抑制剂（抗miR-92a-3p）转染软骨生成的人间充质干细胞（hMSCs）和人软骨细胞。通过酶联免疫吸附测定（ELISA）定量ADAMTS-4/5蛋白产量，通过免疫印迹检测miR-92a-3p参与白细胞介素-1β（IL-1β）介导的分解代谢作用。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（NF）-κB的作用。通过荧光素酶报告基因测定证实miR-92a-3p与其在ADAMTS-4/5 mRNA的3'-非翻译区（3'-UTR）中假定结合位点之间的相互作用。
miR-92a-3p在软骨生成的hMSCs中表达升高，在OA软骨中的表达明显低于正常软骨。IL-1β刺激显著降低原代人软骨细胞（PHCs）中miR-92a-3p的表达。用miR-92a-3p转染软骨细胞可下调IL-1β诱导的ADAMTS-4/5表达，以及含有人类ADAMTS-4/5 mRNA 3'-UTR的报告构建体的活性。在软骨细胞中，IL-1β诱导的MAPK和NF-κB激活后，miR-92a-3p表达受到抑制。
miR-92a-3p是人类软骨细胞中ADAMTS-4/5的重要调节因子，可能有助于OA的发展。
