Effect of RNAi on the expression of COX-2 in human rheumatoid arthritis synovial fibroblasts
- VernacularTitle:RNAi抑制类风湿关节炎成纤维细胞环氧合酶-2合成及对炎症因子的影响
- Author:
Lin-Di JIANG
;
Han-Zhou WANG
;
Feng-Di ZHAO
;
Ru-Yi XUE
;
Jin-Sheng GUO
;
Chun-De BAO
;
Ji-Yao WANG
;
- Publication Type:Journal Article
- Keywords:
Arthritis rheumatoid;
Cyclooxygenase;
Fibroblasts;
Prostaglandin E2;
RNA interfering
- From:
Chinese Journal of Rheumatology
2001;0(05):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To design,synthesize and screen high efficient small interfering RNA(siRNA) targeting to cyclooxygenase-2(COX-2)on rheumatoid arthritis synovial fibroblasts(RASF).To further study the effect of specific COX-2 siRNA interfering on mediators of inflammatory cytokines.Methods Four pairs of siRNA for human COX-2 mRNA were synthesized by utilizing RNA design software,while another random sequence was designed as control.They were divided into group A to H.Among them,group A was used as the negative control(CTL),and group B to F were transfected as random siRNA(NC),1#～4#siRNA in order. These siRNAs were transferred into RASF by LipofectAMINE2000 package and PMA(phorbol-12-myristate- 13-acetate)was added into each culture and with a final concentration of 100 nmol/l.RASF was collected 48 hours after transfection.The expression of hCOX-2 at mRNA level was determined by reverse transcription- polymerase chain reaction(RT-PCR)and hCOX-2 protein level by Western Blot.The supernatant levels of PGE_2,IL-1?,IL-6,TNF-?and vascular endothelial growth factor(VEGF)of the above groups were detected by ELISA.Results The levels of hCOX mRNA and protein in RASF treated with 4-#siRNA were significantly lower than those of the negative control and other groups.The level of PGE_2 and cytokines like IL-1?,IL-6, TNF-?and VEGF in the supernatant were lower in the 4#siRNA group than in other groups.Conclusion 4#siRNA can effectively inhibit the expression of COX-2 mRNA and the synthesis of the COX-2 protein in human synovial fibroblasts.The level of PGE_2,IL-1?,IL-6,TNF-?and VEGF is the lowest in the super- natant.Thus 4#siRNA has been confirmed to specifically block the COX-2 in human synovial fibroblasts.
