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THU0026 microRNA-365 Regulates Expression of Catabolic Factors via Modulating IL-1β-Induced EPAS-1 Expression in SW1353 Cells and Human Primary Chondrocytes
  1. G.H. Park1,
  2. C.M. Lee1,
  3. S.Y. Lee1,
  4. K.M. Son2,
  5. H.S. Lee2,
  6. H.S. Hwang1,
  7. M.H. Lee1,
  8. H.A. Kim1
  1. 1Division of Rheumatology, Department of Internal Medicine, Hallym University Sacred Heart Hospital, Anyang, Kyunggi, 431–070
  2. 2Division of Rheumatology, Department of Internal Medicine, Hallym University Chuncheon Sacred Heart Hospital, Chuncheon, Korea, Republic Of

Abstract

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

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