Background MicroRNAs, small noncoding RNA molecules, are involved in the pathogenesis of various diseases such as cancer and arthritis. Endothelial PAS domain protein-1/hypoxia-inducible factor-2α (EPAS-1/ HIF-2α) is a catabolic transcription factor that regulates osteoarthritis (OA) cartilage destruction.
Objectives In this study, we examined whether microRNA-365 (miR-365) affects interleukin (IL)-1β-induced expression of catabolic factors in chondrocytes via regulation of HIF-2α.
Methods Total RNA was isolated from normal and OA cartilage tissues and human chondrocytes. miR-365 expression was quantified by TaqMan assay. The effects of miR-365 on HIF-2α and HIF-2α-modulated genes were assessed by quantitative real-time reverse polymerase chain reaction (qRT-PCR), Western blot analysis, and enzyme-linked immunosorbent assay (ELISA). Direct interaction of miR-365 with the 3' untranslated region (UTR) of HIF-2α mRNA was examined using a luciferase reporter assay. Nitrite concentration was measured in culture medium using a Griess assay.
Results miR-365 levels were significantly decreased in human OA cartilage relative to normal cartilage. Overexpression of miR-365 significantly suppressed IL-1β-induced expression of HIF-2α in human articular chondrocytes. Pharmacological inhibition of various IL-1β-associated signaling pathways revealed mitogen-activated protein kinase and nuclear factor-κB as the primary pathways driving IL-1β-mediated decreases in miR-365 and subsequent increase in HIF-2α expression. Using a luciferase reporter assay encoding the 3'UTR of human HIF-2α mRNA, we showed that overexpression of miR-365 significantly suppressed IL-1β-induced up-regulation of HIF-2α. 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, and nitric oxide (NO) production in chondrocytes.
Conclusions MiR-365 regulates IL-1β-stimulated catabolic effects in human chondrocytes by modulating HIF-2α expression.
Yang et al. Hypoxia-inducible factor-2alpha is a catabolic regulator of osteoarthritic cartilage destruction, Nat. Med. 16 (2010) 687–693.
Akhtar et al. Haqqi, MicroRNA-27b regulates the expression of matrix metalloproteinase 13 in human osteoarthritis chondrocytes, Arthritis Rheum. 62 (2010) 1361–1371.
Park et al. MicroRNA-558 regulates the expression of cyclooxygenase-2 and IL-1beta-induced catabolic effects in human articular chondrocytes, Osteoarthritis Cartilage 21 (2013) 981–989.
Acknowledgements This study was supported by the Basic Science Research Program through the National Research Foundation (NRF) of Korea funded by the Ministry of Education (2014R1A1A2059823 and 2016R1D1A1B03932259).
Disclosure of Interest None declared