DNA Methylation Status of the IL-8Gene in CD4~+T Cells in Rheumatoid Arthritis
|School||Central South University|
|Keywords||rheumatoid arthritis CD4~+T cells IL-8 methylation|
Objects:Rheumatoid arthritis (RA) is an autoimmune disease involving peripheral joints with multi-systemic inflammatory. Abnormal DNA methylation contributes to the development of rheumatoid arthritis. Interleukin-8(IL-8) plays an important role in inflammatory disease. However, the mechanisms leading to the over-expression of IL-8in RA patients are not well understood. In this study, we investigate the DNA methylation status of regulatory elements of IL-8gene in CD4+T cells of patients with RA and the relationship between DNA methylation levels and expression levels of IL-8gene.Methods:The levels of ESR and CRP were detected by Automatic biochemical analyzer. To extract peripheral blood (60ml) in controls and patients with RA, peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral venous blood of RA patients and healthy donors by density gradient centrifugation. CD4+T cells were isolated using microbeads and protocols provided by the manufacturer. DNA and RNA in CD4+T cell were extracted by kits. Real-time RT-PCR was used to detect the expression of IL-8mRNA. ELISA kit was used to measure IL-8protein concentrations in the serum from RA patients and healthy controls. Bisulfite sequencing was used to determine the methylation status of the regulatory sequence of IL-8gene. Results:Compared to negative controls, the expression of IL-8mRNA were significantly elevated in patients (P=0.028). ELISAs showed serum protein levels were significantly increased in RA patients (P﹤0.01). Bisulfite sequencing results showed the DNA methylation level of the regulatory sequence of IL-8gene was significantly lower in RA CD4+T cells than in controls (P=0.038), it was inversely correlated with the IL-8mRNA expression (P=0.024,r=-0.492).Conclusions::(1)The expression of IL-8mRNA were significantly elevated in patients with RA;(2)Serum protein levels were significantly increased in RA patients;(3)DNA Hypomethylation contributes to the overexpression of IL-8in CD4+T cells of RA patients. Aberrant DNA methylation modification plays an important role in the pathogenesis of RA.