Objectives Endothelial PAS domain protein-1 (EPAS-1/HIF-2α), encoded by EPAS1 gene, is a catabolic transcription factor to regulate osteoarthritis (OA) cartilage destruction. In this study we examined whether microRNA-365 (miR-365) can regulate interleukin (IL)-1β-induced expression of catabolic factors in SW1353 cells and human articular chondrocytes via regulation of EPAS-1.
Methods Total RNA was isolated from normal and OA cartilage tissues, human chondrocytes, and SW1353 cells. The level of miR-365 was quantified by TaqMan assay. The effects of miR-365 on expression of EPAS-1 and EPAS-1-modulated genes were measured by quantitative real-time polymerase chain reaction (qRT PCR), Western blot analysis, and enzyme-linked immunosorbent assay (ELISA). To examine the direct interaction of miR-365 with 3' untranslated region (UTR) of EPAS-1 mRNA, luciferase reporter assay was performed.
Results The level of miR-365 in human OA cartilage was significantly decreased compared to normal cartilage. The overexpression of miR-365 significantly suppressed IL-1β-induced expression of EPAS-1 in both SW1353 cells and human articular chondrocytes. QRT PCR analysis using pharmacological inhibitors revealed that mitogen activated protein kinase and nuclear factor-κB signaling pathway was involved in IL-1β-induced miR-365 decrease and subsequent increase of EPAS-1 expression. Luciferase reporter assay using DNA construct containing binding site of miR-365 within 3'UTR of human EPAS-1 mRNA demonstrated that the overexpression of miR-365 significantly suppressed IL-1β-induced up-regulation of EPAS-1. Furthermore, miR-365 overexpression significantly suppressed IL-1β-induced expression of catabolic factors, including cyclooxygenase-2, inducible nitric oxide synthase, and matrix metalloproteinase-1, -3 and -13, in SW1353 cells and human chondrocytes.
Conclusions MiR-365 regulates IL-1β-stimulated catabolic effects in SW1353 cells and human chondrocytes via modulating the expression of EPAS-1.
Disclosure of Interest None declared