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

Objective To screen highly efficient small interfering RNA(siRNA)targeting human cyelooxygenase-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-1β(IL-1 β), interleukin-6(IL-6),timorous necrosis faetor-α(TNF-α),and vascular endothelial growth factor (VEGF).Methods 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).Phorbo]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-1β,IL-6,TNF-α,and VEGF were measured by ELISA.Results 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-1β,IL-6,TNF-α,and VEGF in the culture supernatant were lower in the 4~# siRNA group than in other groups 24,36,and 48 h after transfeetion.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.Conclusion 4~#siRNA effectively inhibits the expression of COX-2 mRNA and protein the level of PGE2,IL-1β,IL-6,TNF-α,and VEGF in the clear supematant of the 4~#group is lowest.