uPAR在类风湿关节炎成纤维样滑膜细胞中的功能及机制

Effects and Mechanisms of uPAR Expression in Rheumatoid Arthritis Fibroblast-like Synoviocytes

【目的】采用RNA干扰(RNAi)技术阻断类风湿关节炎(RA)成纤维样滑膜细胞(FLS)尿激酶型纤溶酶原激活物受体(uPAR)基因的表达,探讨uPAR基因沉默后对RA-FLS功能产生的影响及有关机制。【方法】收集行关节置换术或滑膜清理术的RA患者的滑膜组织,组织块法培养RA-FLS。通过阳离子脂质体转染的方法,转染体外合成的特异性uPARsiRNA;利用荧光定量PCR法和Western blotting法检测uPAR沉默效果;CCK8法检测细胞增殖抑制率;流式细胞技术检测细胞周期改变;Transwell趋化小室测定细胞的迁移能力;Western blotting技术分析沉默uPAR基因对RA-FLS中PI3K/AKT通路的影响。【结果】uPAR-siRNA能有效阻断RA-FLS中uPAR基因在mRNA和蛋白水平上的表达。沉默uPAR基因后,细胞48、72、96 h增殖抑制率分别为(17.51±2.27)%、(28.62±4.82)%、(22.91±5.78)%,显著高于对照组(P<0.05);uPARsiRNA干扰后,被阻滞于G0/G1期的细胞增加,而S和G2/M期的细胞减少;Transwell趋化小室结果显示:与对照组相比,特异性干扰组的RA-FLS迁移细胞数(35±11)相比空白对照组(138±21)和NC-siRNA组(136±19)明显减少(P<0.05);转染后的RA-FLS相对于对照组,PI3K、AKT、GSK3β的磷酸化水平显著降低(P<0.05)。【结论】uPAR在RA-FLS的增殖、周期及迁移中起重要作用,这可能与其对PI3KAKT通路的激活有关。

【Objective】 To observe the effects and mechanism of urokinase-type plasminogen activator receptor(uPAR) expression in rheumatoid arthritis fibroblast-like synoviocytes(RA-FLS).【Methods】 Tissues were collected from RA patients with joint replacement surgery or arthroscopy and RA-FLS were obtained by tissue culture.Chemically synthesized small interference RNA(siRNA) specifically targeting uPAR gene was transfected into RA-FLS by cationic liposome.The interference efficiency of uPARsiRNA on the production of uPAR mRNA and protein was determined by RT-qPCR and Western blotting respectly.The proliferative inhibition rate was examined by CCK8 assay.Flow cytometry was adopted to determine the change of cell cycle distribution.The migration of RA-FLS was examined by Transwell assay.Western blotting was performed to detect the influence of uPAR on PI3K/AKT signal pathway.【Results】 Transfection of uPAR-siRNA significantly decreased the mRNA and protein expression of uPAR gene.The proliferative inhibition rate was obviously higher in the uPAR-siRNA group than the control groups(P < 0.05) after transfection for 48 h(17.51 ± 2.27)%,72 h(28.62 ± 4.82)%,96 h(22.91 ± 5.78)%.Flow cytometry assay showed accumulation of cells in the G0/G1 phase and the number of RA-FLS in the S and G2/M decreased;Transwell migration assay demonstrated the RA-FLS through the transwell membrane in uPAR-siRNA group(35 ± 11) were lesser than the NC-siRNA group(136 ± 19)(P < 0.05) and the blank control group(138 ± 21)(P < 0.05).After transfected with uPAR-siRNA,the phosphorylation of PI3K/AKT/GSK3β decreased significantly.【Conclusion】 uPAR may play a role in the regulation of proliferation,cell cycle and migration of RA-FLS through activation of PI3K/AKT/GSK3β signal pathway.