Demethylation of MicroRNA-124a Genes Attenuated Proliferation of Rheumatoid Arthritis Derived Fibroblast-Like Synoviocytes and Synthesis of Tumor Necrosis Factor-α

Abstract

Objective: To examine the impact of 5-Aza-2'-deoxycytidine (5-AzadC) on methylation status of miR-124a genes in rheumatoid arthritis (RA) associated fibroblast-like synoviocytes (FLS) and its effect on RA-FLS proliferation and TNF-α expression.

Materials and methods: FLS were isolated from seven RA-derived synovial tissues and cultured in vitro. The expression of miR-124a was measured by real time quantitative polymerase chain reaction (PCR) in FLS with or without 5-AzadC treatment. MiR-124a gene methylation was detected by methylation-specific PCR. FLS were divided into three groups as control, IL-1β and IL-1β/5-AzadC, respectively. The cells in the IL-1β group were treated with 5 μg/L of IL-1β for 24 hours, whereas the cells in the IL-1β/5-AzadC group were first treated with IL-1β exactly as those in the IL-1β group for 24 h but further treated with 1μM 5-AzadC for additional 3 days. The cell growth was estimated based on absorbance at UV450nm. Secreted TNF-α from the cells was evaluated by enzyme-linked immunosorbent assay. After that, RA-FLS treated with IL-1β plus 5-AzadC were further transfected with miR-124a inhibitor or scrambled control. After culturing for 3 days, cell growth and TNF-α concentrations were measured.

Results: After 5-AzadC treatment, the expression of miR-124a was significantly increased compared with the control group (1.545 ± 0.189 vs 0.836 ± 0.166, p = 0.001). On the other hand, 5-AzadC significantly reduced IL-1β-mediated cell proliferation by nearly 2.5 fold (p = 0.006). Also, the level of TNF-α secreted from the cells treated with IL-1β plus 5-AzadC was considerably less than that from the cells treated with IL-1β alone (324.99 ± 22.73 ng/L vs 387.91 ± 58.51 ng/L, p = 0.022). After transfection with miR-124a inhibitor in RA-FLS treated with IL-1β plus 5-AzadC, the cell proliferation was increased by 18.2% and the TNF-α expression was increased by 19.0% (p = 0.001 and 0.011, respectively).

Conclusion: Methylation of miR-124a genes contributed to IL-1β-mediated RA-FLS proliferation and TNF-α expression.

Fig 1. Analysis of miR-124a expression.

FLS were isolated from 7 RA patients, cultured in vitro, and treated with 5-AzadC for 3 days. Expression of miR-124a in the treated cells was analyzed by real time quantitative PCR and represented either on the average (left) or individually (right).

Fig 2. 5-AzadC reduced IL-1β-mediated cell proliferation.

FLS were treated with 5 ng/mL IL-1β alone for 4 days, or with IL-1β for 24 h and then with 1 μM 5-AzadC for 3 days, or without any treatments (control). Cell proliferation was estimated as described in the Materials and Methods. Cell growth was represented either individually (left) or on the average(right). ^#, comparison with the IL-1β group, p< 0.05.

Fig 3. Analysis of TNF-α expression.

FLS were treated exactly as described in the legend for Fig 2. The conditioned media were collected from each group, and the concentration of TNF-α in the conditioned media was estimated by ELISA. *, comparison with the control group, p<0.05; ^#, comparison with the IL-1β group, p< 0.05.