Effects of shRNA-IL-32γ-mediated gene silencing on the proliferation and apoptosis of fibroblast-like synoviocytes in patients with rheumatoid arthritis / 中华风湿病学杂志

ABSTRACT

ObjectiveTo investigate the effects and mechanisms of IL-32γ-shRNA-mediated gene silencing on the proliferation and apoptosis of fibroblast-like synoviocytes in patients with rheumatoid arthritis (RA).MethodsA eukaryotic expression plasmid of shRNA targeting IL-32γ was transfected into fibroblastlike synoviocytes by liposome in patients with rheumatoid arthritis.RT-PCR was used to determine the expression level of IL-32γ.Western blotting was used to detect the levels of cyclin D1 and p-Akt.The proliferation of RA-FLS was examined by MTT.Cell cycles were analyzed by flow-cytometry.The apoptosis of cells were measured by TUNNEL.Comparisons between groups were tested by t test.Results ① The expression of IL-32γwas significantly inhibited by shRNA-IL-32γ-expressing plamid PGCsi 3.0 targeting sequence 1,2 and 3,and the inhibition rate had reached 75.6％,66.2％ and 64.1％,respectively.② The absorbance value of proliferation of RA-FLS in EASY-shRNA-IL-32γ group was significantly lower than that in the shRNA-control group and normal group at day 3 [(0.23±0.03) vs (0.35±0.03) and (0.36±0.04),P＜0.05] and 5 [(0.27±0.03) vs (0.52±0.05) and (0.53±0.04),P＜0.01 ] after transfection.③ The rate of RA-FLS at phase G1 in the EASY-shRNA-IL-32γ group was significantly higher than that in the shRNA-control group and normal group respectively [(88±6)％ vs (69±5)％ and (68±4)％,P＜0.05],while those at phase S+G2 in the EASY-shRNA-IL-32γgroup was significantly lower than that in the shRNA-control group and normal group [ ( 13.6±3.0)％ vs (30.2±4.1)％ and(32.1±4.3)％,P＜0.01].④The rate of RA-FLS apoptosis in the EASY-shRNA-IL-32γ group was significantly higher than that in the shRNA-control group and normal group[(20.50±3.21 )％ vs (9.20±0.32)％ and (8.60±0.22)％,P＜0.01].⑤ The expression of cyclin D1(0.36±0.04) and p-Akt(0.31±0.03) in the EASY-shRNA-IL-32γ group was significantly lower than that in the shRNA-control group [ (0.59±0.08) and (0.53±0.06)] and normal group [(0.61±0.07) and (0.52±0.06),P＜0.01].ConclusionEASYshRNA-IL-32γ can inhibit RA-FLS proliferation by down-regulating the expression of cyclin D1 and induce RA-FLS apoptosis by down-regulating the expression of p-Akt.