Effects Of ShRNA-HIF-1α-mediated Gene Silencing On Expression Of VEGF At Fibroblast-like Synoviocytes And Its Proliferation In Patients With Rheumatoid Arthritis

Objective: To investigate the expression of hypoxia-inducible factor-1α and vascularendothelial growth factor on fibroblast-like synoviocytes cells in rheumatoid arthritis and itscorrelation at normoxia and hypoxia. Meanwhile to investigate the effects of shRNA-HIF-1α-mediated gene silencing on expression of VEGF at fibroblast-like synoviocytes(FLS) and itsproliferation in patients with rheumatoid arthritis in vitro.Methods:1. Primary cultured fibroblast-like synoviocytes cells form RA and OA patients wereexposed to normoxia and hypoxia for6,12hours. The expression of HIF-1α and VEGF weredetected by Western blot.2. The human synovial tissue from rheumatoid arthritis patients were cultured and passed. TheFLS were divided into4groups: hypoxic group, normoxic group, the negative control group (withFLS blank plasmids) and the positive group (with FLS HIF-1α-shRNA).The hypoxia group cellswere incubated under normoxia. The last three groups’ cells were incubated under hypoxia. Theexpression of HIF-1α and VEGF protein and mRNA were detected by Western blot and real-timePCR. The normoxia group, the negative control group and the positive group cells were incubatedunder hypoxia for6,12,24and48hours. The proliferation of RA-FLS was examined by MTT.Results:1. Higher expressions of HIF-1α and VEGF of hypoxia6h and12h were detected infibroblast-like synoviocytes cells form RA and OA patients compared with those in normoxiagroup(P<0.05). Compared with hypoxia6h group, the expression of HIF-1α and VEGF of hypoxia12h group were higher on fibroblast-like synoviocytes between RA and OA (P<0.05). Theexpression of HIF-1α was positively correlated to VEGF in three groups (P<0.05）. Comparedwith the expression of HIF-1α and VEGF in fibroblast-like synoviocytes cells of RA and OApatients：there were no significant difference in normoxic group(P>0.05), but the expression ofHIF-1α and VEGF in6h and12h hypoxia group of fibroblast-like synoviocytes cells in RApatients were higher than that of the same group of OA patients(P<0.05). 2. Higher expressions of HIF-1α and VEGF protein and mRNA on FLS were detected inhypoxic group compared with those in normoxia group (P<0.05). The expression of HIF-1α andVEGF protein and mRNA were no significant differences between the normoxic group and thenegative control group (P>0.05). Compared with the normoxic group and the negative controlgroup, the expression of HIF-1α and VEGF protein and mRNA of the positive group weresignificantly down-regulated (P<0.05). The expression of HIF-1α were positively correlated toVEGF in four groups (P<0.05).MTT assays showed that the FLS proliferation of the positivegroup were slower than that of the normoxic group and the negative control group (P<0.05).Conclusions:1. The expressions of HIF-1α and VEGF on fibroblast-like synoviocytes cells aresignificantly higher in RA than that in OA.“Hypoxia-HIF-1α-VEGF” signaling pathway may beclosely related to the occurrence and development of RA.2. The plasmids of HIF-1α-shRNA can inhibit the expression of HIF-1α and VEGF proteinand mRNA in RA-FLS and inhibit the FLS proliferation significantly.