Role of DNA methylation on 15-Lipoxygenase-1 gene expression in Osteoarthritis

Abstract:
Objective and design. Epigenetic changes particularly DNA methylation plays an essential role in the regulation of gene expression. DNA methylation was reported to control the expression of several genes involved in the pathophysiology of OA including genes encoding for the pro-inflammatory mediators and the cartilage degrading enzymes. Numerous anti-inflammatory and immunomodulatory lipid mediators are produced by 15-lipoxygenase-1 (15-LOX-1), which has been shown to have protective effects against a number of inflammatory diseases, including OA. The purpose of this study was to assess the expression of 15-LOX-1 in the cartilage of OA patients and normal donors, as well as to ascertain whether DNA methylation controls this expression. Methods. Cartilage samples were collected from both OA-affected at the time of total knee replacement surgery, and normal knee joints during autopsy. Real-time polymerase chain reaction (PCR) was used to assess the expression of 15-LOX-1. Using 5-Aza-2'-desoxycytidine (5-Aza-dC), a DNA methyltransferase inhibitor, the significance of DNA methylation in 15-LOX-1 expression was evaluated. Using a CpG-free luciferase vector, the impact of CpG methylation on the activity of the 15-LOX-1 promoter was assessed. Pyrosequencing was used to ascertain the 15-LOX-1 promoter's DNA methylation status. Results. Compared to healthy cartilage, OA showed increased expression of 15-LOX-1. 15-LOX-1 mRNA levels were upregulated in chondrocytes treated with 5-Aza-dc, and 15-LOX-1 promoter activity was downregulated in vitro through methylation. The methylation status of the 15-LOX-1 gene promoter did not differ between cartilage from normal and OA cartilage. Conclusion. In OA cartilage, there was an increased expression level of 15-LOX-1, which might be related to a healing process. The methylation status of 15-LOX-1's promoter was not linked to its upregulation in OA cartilage, indicating that alternative mechanisms may be at play.