Expression of miRNA-146a in the inflammatory response of RAW264.7 cell through Toll-like receptor signaling pathway in gout arthritis model / 中华风湿病学杂志

ABSTRACT

Objective:To investigate the possible role of miR-146a in the patho-genesis of inflammation in primary gout arthritis.Methods:① The RAW264.7 mouse macrophage was stimulated with 200 μg/ml monosodium urate (MSU) crystal for 0 h, 3 h, 6 h, and 12 h. Then cells and super-natants were collected. The miR-146a was detected by TaqMan probe method. The expression of interleukin-1 receptor-associated kinase 1 (IRAK 1), tumor necrosis factor receptor-associated factor 6 (TRAF6), nuclear factor-kappa B (NF-κB), interleukin (IL)-1β, tumor necrosis factor-α (TNF-α) mRNA were detected by real-time (RT)-quantitative polymerase chain reaction (qPCR). The concentration of IL-1β was measured in the culture supernatant by enzyme-linked immunosorbent assay (ELISA). The protein expression levels of TRAF6, NF-κB and IL-1β were detected by Western-blotting. ② The RAW264.7 mouse macrophage was transfected with miR-146a mimics, miR-146a mimic control, miR-146a inhibitor, and miR-146a inhibitor control. After stimulating each group of cells with 200 μg/ml MSU crystals for 6 h, the expression of miR-146a, IRAK1, TRAF6, NF-κB, IL-1β mRNA and TRAF6, NF-κB, IL-1β protein were measured. The measurement data were compared by Independent sample t test. and repeated measures analysis of variance (ANOVA). Results:① After MSU crystals stimulated RAW264.7 cells, we found that the expression level of miR-146a in the stimulation group at 3 h, 6 h, and 12 h was lower than that in the control group ( t=-10.234, -17.059, -26.204, P<0.01), and then, IL-1β protein concentration at 6 h, 12 h was higher ( t=7.552, 9.007, P<0.01). Meanwhile, IRAK1, TRAF6, NF-κB and IL-1β mRNA in the stimulation group at 3 h and 6 h were higher than those in the control group ( t=9.847, 6.147, P<0.01; t=3.49, 3.32, P<0.05; t=3.643, 8.471, P<0.05; t=8.726, 49.68, P<0.01). TNF-α mRNA at three time points in the stimulation group was high ( t=4.691, 11.115, 12.816, P<0.01). Moreover, the results showed that the relative expression of TRAF6 and NF-κB protein in the stimulation group at 6 h and 12 h was higher than that in the control group ( t=8.052, 8.119, P<0.01, t=22.454, 5.845, P<0.01), IL-1β protein in the stimulation group increased at all three time points compared with the control group ( t=18.561, 4.74, 8.432, P<0.01). ② After trans fection, the miR-146a mRNA expression of the mimics group was significantly higher than the mimics control group ( t=31.769, P<0.01); the inhibitor group was significantly lower than the inhibitor control group ( t=-4.22, P<0.05). ③miR -146a overexpression group was stimulated with 200 μg/ml MSU crystals for6 h, the expression levels of IRAK 1, TRAF 6, NF-κB and IL-1β mRNA in the mimic group were lower than those in the mimic control group ( t=-14.754, -21.201, -19.381, -17.323, P<0.01), the expression levels of TRAF 6, NF-κB and IL-1β protein were also lower than those in the mimic control group ( t=-3.137, -32.974, -18.789, P<0.05), while the inhibitor group had good results. Conclusion:① Overexpression of miR-146a can reduce the expression of IRAK1, TRAF6, NF-κB, IL-1β and inhibit MSU crystal-mediated inflammation, while inhibition of miR-146a expression can aggravate inflammation, suggesting that miR-146a participates in the negative feedback regulation of gout inflammation. ② miR-146a may target the NF-κB signaling pathway and participate in spontaneous remission of gouty arthritis.