[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 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).


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). 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.


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-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.


CONCLUSIONS
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.
Jiang
Lin-di
LD
Department of Rheumatology, Zhongshan Hospital, Fudan University, Shanghai 200032, China.
Wang
Han-zhou
HZ
Zhao
Feng-di
FD
Guo
Jin-sheng
JS
Bao
Chun-de
CD
Zou
He-jian
HJ
Wang
Ji-yao
JY
D001172:Arthritis, Rheumatoid / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D015153:Blotting, Western; D051546:Cyclooxygenase 2 / Q000235:genetics* / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D007375:Interleukin-1 / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013583:Synovial Membrane / Q000378:metabolism / Q000473:pathology*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D007375:Interleukin-1; D053583:Interleukin-1beta; D015850:Interleukin-6; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D042461:Vascular Endothelial Growth Factor A; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D015232:Dinoprostone