Silencing of microRNA-138-5p promotes IL-1β-induced cartilage degradation in human chondrocytes by targeting FOXC1: miR-138 promotes cartilage degradation

Abstract

Objectives: Osteoarthritis (OA) is characterised by articular cartilage degradation. MicroRNAs (miRNAs) have been identified in the development of OA. The purpose of our study was to explore the functional role and underlying mechanism of miR-138-5p in interleukin-1 beta (IL-1β)-induced extracellular matrix (ECM) degradation of OA cartilage.

Materials and methods: Human articular cartilage was obtained from patients with and without OA, and chondrocytes were isolated and stimulated by IL-1β. The expression levels of miR-138-5p in cartilage and chondrocytes were both determined. After transfection with miR-138-5p mimics, allele-specific oligonucleotide (ASO)-miR-138-5p, or their negative controls, the messenger RNA (mRNA) levels of aggrecan (ACAN), collagen type II and alpha 1 (COL2A1), the protein levels of glycosaminoglycans (GAGs), and both the mRNA and protein levels of matrix metalloproteinase (MMP)-13 were evaluated. Luciferase reporter assay, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot were performed to explore whether Forkhead Box C1 (FOCX1) was a target of miR-138-5p. Further, we co-transfected OA chondrocytes with miR-138-5p mimics and pcDNA3.1 (+)-FOXC1 and then stimulated with IL-1β to determine whether miR-138-5p-mediated IL-1β-induced cartilage matrix degradation resulted from targeting FOXC1.

Results: MiR-138-5p was significantly increased in OA cartilage and in chondrocytes in response to IL-1β-stimulation. Overexpression of miR-138-5p significantly increased the IL-1β-induced downregulation of COL2A1, ACAN, and GAGs, and increased the IL-1β-induced over expression of MMP-13.We found that FOXC1 is directly regulated by miR-138-5p. Additionally, co-transfection with miR-138-5p mimics and pcDNA3.1 (+)-FOXC1 resulted in higher levels of COL2A1, ACAN, and GAGs, but lower levels of MMP-13.

Conclusion: miR-138-5p promotes IL-1β-induced cartilage degradation in human chondrocytes, possibly by targeting FOXC1.Cite this article: Y. Yuan, G. Q. Zhang, W. Chai,M. Ni, C. Xu, J. Y. Chen. Silencing of microRNA-138-5p promotes IL-1β-induced cartilage degradation in human chondrocytes by targeting FOXC1: miR-138 promotes cartilage degradation. Bone Joint Res 2016;5:523-530. DOI: 10.1302/2046-3758.510.BJR-2016-0074.R2.

Keywords: FOXC1; IL-1β; MicroRNA-138; cartilage degradation; osteoarthritis.

miR-138-5p is upregulated in OA cartilage and in chondrocytes in response to IL-1β; a) relative expression of miR-138-5p in normal and OA cartilage; b) relative expression of miR-138-5p in normal and OA chondrocytes; c) relative expression of miR-138-5p stimulated by IL-1β. (miR, microRNA; OA, osteoarthritis; IL-1β, interleukin-1 beta.) ^* p < 0.05; ^** p < 0.01.

Alteration of miR-138-5p affects IL-1β-induced anabolic and catabolic metabolism in OA chondrocytes. The cells in the control group were maintained in culture media only, and the cells in other groups were treated with IL-1β alone or transfected with miR-138-5p mimics or ASO-miR-138-5p: a) relative expression of COL2A1; b) relative expression of ACAN; c) relative expression of GAGs; d) protein expression of MMP-13; e) relative mRNA expression levels of MMP-13. (miR-138, microRNA-138; OA, osteoarthritis; IL-1β, interleukin-1 beta; COL2A1, collagen, type II, alpha 1; ACAN, aggrecan; GAGs, glycosaminoglycans; MMP, matrix metalloproteinase; ASO, allele-specific oligonucleotide.) *p < 0.05; **p < 0.01.

FOXC1 is a direct target of miR-138-5p and is negatively regulated by miR-138-5p. The cells in the control group were transfected with miR-138-5p mimics or ASO-miR-138-5p negative controls, and the cells in other groups were transfected with miR-138-5p mimics or ASO-miR-138-5p: a) software prediction of miR-138-5p potential binding sites on FOXC1 3’-UTR; b) relative luciferase activity in co-transfection of miR-138-5p mimics with WT or MUT-FOXC1 3’-UTR vectors; c) relative luciferase activity in co-transfection of ASO-miR-138-5p with WT or MUT-FOXC1 3’-UTR vectors; d) relative mRNA levels of FOXC1 after alteration of the expression of miR-138-5p; e) relative protein levels of FOXC1 after alteration of the expression of miR-138-5p; f) representative pictures of Western blot. (miR-138, microRNA-138; OA, osteoarthritis; FOXC1, Forkhead Box C1; UTR, untranslated regions; WT, wild type; MUT, mutant; ASO, allele-specific oligonucleotide; NC, negative control; NS, no significance.) ^*p < 0.05;^**p < 0.01.

FOXC1 reverses the effects of miR-138-5p-mediated IL-1β-induced matrix degradation. The cells in the control group were maintained in culture media only, and the cells in other groups were treated with IL-1β alone or with miR-138-5p mimics or combined with pcDNA3.1 (+)-FOXC1. a) relative expression of COL2A1; b) relative expression of ACAN; c) relative expression of GAGs; d) protein expression of MMP-13; e) relative mRNA expression levels of MMP-13. (miR-138, microRNA-138; FOXC1, Forkhead Box C1; IL-1β, interleukin-1 beta; COL2A1, collagen, type II, alpha 1; ACAN, aggrecan; GAGs, glycosaminoglycans; MMP, matrix metalloproteinase; ASO, allele-specific oligonucleotide.) ^*p < 0.05; ^**p < 0.01.