| BACKGROUNDRheumatoid arthritis (RA) is one of the common chronic auto-immune diseases involved in small joints of the limbs such as the hands and feet. It would cause articular destruction, malformation and disability and eventually might lead to labor loss and early death. The cause of the disease is still unclear so far. Substantial initiation triggers inflammatory synovium of RA. Then the membrane phospholipid breaks down into arachidonic acid. With the promotion of a series of enzymes such as cyclooxygenase (COX), pre-mediators of inflammation like prostaglandin E2 (PGE2), and cytokines like interleukin-1β(IL-1β), interleukin-6 (IL-6) and tumorous necrosis factor-a (TNF-a) are synthesized. COX is the important rate-limiting enzyme during the transformation from arachidonic acid to prostaglandin. Studies have confirmed the increase of the COX-2 expression in the synovial tissues of rheumatoid arthritis patients. The activity of COX-2 is directly related to the amount of prostaglandin product in the articular tissues, which might play an important role in the initiation and promotion of arthritis. PGE2 has multiple biological functions, including promotion of articular inflammation, degradation of the cartilage and increasing the synthesis of matrix metalloproteinase (MMP). COX-2 is also the key during the process of vascularization on the inflamed synovium. The non-steroidal anti-inflammatory drugs (NSAIDs) which are firstly used in the treatment of RA present the effects of anti-inflammation and pain relieving, by inhibiting the COX activity and blocking the synthesis of prostaglandin. However, they also might induce many adverse effects, such as inhibiting platelet function, reducing renal blood flow, causing liver toxicity and injuring the gastrointestinal tract, among which serious events including ulcer, bleeding or perforation of the gastrointestinal tract might occur and lead to admission or poor consequence. Therefore, our research tried to find the efficient small interfering RNA (siRNA), which could specifically inhibit the COX-2 gene expression and protein production in human rheumatoid arthritis synovial fibroblasts by the technology of the RNA interfering (RNAi). The functions of the COX-2 gene were investigated in the level of cytology and the growth of synovial fibroblasts of each group was observed.MAIN OBJECTIVETo first design small interfering RNA targeting at human cyclooxygenase-2 (COX-2siRNA) in vitro, and transfect it into human rheumatoid arthritis synovial fibroblast cell, in order to identify the effective siRNA which could specifically block the COX-2 production. Small interfering RNA was transfected to rheumatoid arthritis synovial fibroblasts cell and the mRNA and protein levels of hCOX-2 were measured by RT-PCR and Western Blot respectively.SECONDARY OBJECTIVETo further study the impact of specially interfering COX-2 siRNA on inflammatory mediators (PGE2) and cytokines (IL-1,U IL-6 , TNF-α). To find out its function to vascular endothelial growth factor (VEGF), and thus exploring a new approach to lessening the progression of RA and destruction of the joints. To evaluate the safety of RNAi technology in transfecting C0X-2siRNA into human rheumatoid arthritis synovial fibroblast cell.METHODSThe synovial membrane tissue was taken from defined RA patients met the diagnosed criteria of 1987 ARA undergoing synovectomy. The synovium was isolated, digested, cultured, passed and reserved in liquid nitrogen. According to the characters of the whole gene sequence of COX-2mRNA, the suggestions of published literatures and software provided by public websites, 4 lines of COX-2 mRNA siRNA (1#～4# siRNA), and a scrambled line as control were designed and synthesized. They were separated into 8 different treated groups denominated as group A to group H and corresponding treated agents were arranged the blank control as group A, random siRNA (NC), l#siRNA, 2#siRNA, 3#siRNA, and 4#siRNA in order as group B to group F. Group G and group H were respectively added with 100μmol/L and 500μmol/L of NS-398, one of the selective COX-2 inhibitor. Hela cell was used as positive control. After resuscitation, the human rheumatoid arthritis synovial fibroblast cells were added to the six-hole board, and then different siRNA transfected by LipofectAMINE 2000 kit respectively. 4 hours later, phorbol ester with a final concentration of 200nmol/L was added into each culture. The cells were collected 48 hours after transfection, and the proteins and total RNA of COX-2 were extracted. The expression level of hCOX-2 mRNA was determined by RT-PCR .The expression level of hCOX-2 protein was assessed by Western Blot. The level of the downstream product of PGE2 was measured by ELISA. The inflammatory cytokines like IL-1β, IL-6, TNF-a and VEGF were also measured by ELISA 24h, 36h and 48h after transfection of siRNA.RESULTS1. The level of COXmRNA measured by RT-PCR #4siRNA could significantly knockdown the expression of hCOX-2 than blank control (negative controlled group, CTL), scrambled siRNA group,1#siRNA,2# RNA, 3# RNA group, Hela group and 100μmol/L NS398 group.The ratios of each positively interfering group or Hela group to CTL group were 0.72,0.3, 0.25, 0.4, 0.04, 2.1.and the ratio of each positively interfering group and 100μmol/L or 500μmol/L NS398 group to CTL group 0.71, 0.19, 0.16, 0.25, 0.04, 2.05 and 0.63 respectively estimated by semi-quantitative RT-PCR. It was indicated that the expression of hCOX at mRNA level in 4# siRNA group can remarkably inhibited by 4# siRNA.2. The expression of hCOX-2 protein determined by Western Blot The level of hCOX-2 protein was showed to be significantly lower in the 4# siRNA group than the other groups which was consistent with the result by RT-PCR. The ratios of each positively interfering group (1#siRNA, 2#RNA, 3#RNA,4# group and Hela cell group respectively )to CTL group were 1.04, 0.52, 0.39, 0.9,0 和 2.48 estimated by semi-quantitative method after 36h of transfection and the ratios of each positively interfering group (1#siRNA, 2#RNA, 3#RNA and 4# group respectively ) to CTL group were 1.05, 0.52, 0.51, 0.9, 0.15 after 48h of transfection. It was indicated that the expression of hCOX protein in 4# siRNA group was significantly lower than that of other groups3. The level of PGE2 measured by ELISA The results showed the level of PGE2 inthe culture supernatant of rheumatoid arthritis synovial fibroblast cell was lower in the 4#siRNA group than in the other groups 24h, 36h and 48h respectively after transfection.4. The levels of IL-1β, IL-6, TNF-a and VEGF detected by ELISAThe levels of IL-1β, IL-6, TNF-a and VEGF respectively in the 4#siRNA group decreased markedly among the CTL and other siRNA interfering groups. The values of 4#siRNA group were lower than those of 100μmol/L group 24h, 36h, 48h after transfection and were similar to those of 500μmol/L NS398 group 36h and 48h after transfection respectively.5. Effect of siRNA to the relation between the expression of COX-2mRNA and the levels of PGE2, IL-1β, TNF-α and VEGF The supernatant fluid of the human rheumatoid arthritis synovial fibroblast cell culture in all groups treated by siRNA presented low levels of PGE2, IL-1β, TNF-α and VEGF along with the decrease of COX-2 mRNA expression 24h, 36h and 48h after transfection. It indicated that expression of COX-2 mRNA was parallel to the levels of PGE2, IL-β, TNF-α and VEGF in different groups treated by siRNA.6. The death rate of rheumatoid arthritis synovial fibroblasts The death rates of rheumatoid arthritis synovial fibroblasts among all trial groups measured by trypan blue dye were within the range of 9%～11% and there were not significant difference between CTL group and each siRNAgroup. The proliferation of human rheumatoid arthritis synovial fibroblasts cell transfected with different siRNA from the aspect of morphology and the deathrate did not changed significantly compared 4#siRNA group with other siRNA group or with NS398 group.CONCLUSIONOur study showed that 4#siRNA targeted COX-2 mRNA (CGTTCGACTGAACTGTAGA) could efficiently interrupt the hCOX-2 mRNA expression and protein synthesis in human RA synovial fibroblast cell. The levels of PGE2 and cytokines such as IL-1β, IL-6, TNF-a and VEGE on culture supernatant which play important roles in the RA progression were also found to decrease markedly in the 4#siRNA group. The side-effect of proliferation on growing human synovial fibroblast cell treated by siRNA was not found .
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