Effects of RNAi on cyclooxygenase-2 expression and biologic activity of human rheumatoid arthritis synovial fibroblasts

Abstract

To screen highly efficient small interfering RNA (siRNA) targeting human cyclooxygenase-2 (hCOX-2) mRNA on human rheumatoid arthritis synovial fibroblasts (RASF) and to further study the impact there on prostaglandin E2 (PGE2) and cytokines, such as interleukin-1beta (IL-1beta), interleukin-6 (IL-6), timorous necrosis factor-alpha (TNF-alpha), and vascular endothelial growth factor (VEGF). 4 lines of COX-2 mRNA siRNA (1(#) - 4(#) siRNA) were designed and transfected into the fibroblasts from the synovial membrane of a patient with rheumatoid arthritis (RASF). Phorbol ester was added 4 hours later. A scrambled line (NC) group and a blank control (CTL) group were used. RT-PCR and Western blot ting were used to detect the mRNA and protein expression of COX-2 36 and 48 hours after transfection. The levels of PGE2, IL-1beta, IL-6, TNF-alpha, and VEGF were measured by ELISA. RT-PCR showed that the absorbance ratios of the positively interfering groups, NC, and 1(#) - 3(#) siRNA groups, to CTL group were 0.72, 0.3, 0.25, 0.4, and 0.04 respectively. The ratios of the positively interfering groups to CTL group were 1.04, 0.52, 0.39, 0.9, and 36 h after transfection and 1.05, 0.52, 0.51, 0.9, and 0.15 respectively 48 h after transfection. The levels of PGE2, IL-1beta, IL-6, TNF-alpha, and VEGF in the culture supernatant were lower in the 4(#) siRNA group than in other groups 24, 36, and 48 h after transfection. The death rates of RASF of all trial groups were within the range of 9% - 11% and there were not statistically significant differences between the CTL group and the siRNA groups or between the 4(#) siRNA and other siRNA groups. 4(#) siRNA effectively inhibits the expression of COX-2 mRNA and protein the level of PGE2, IL-1beta, IL-6, TNF-alpha, and VEGF in the clear supernatant of the 4(#) group is lowest.