microRNA-448对类风湿关节炎滑膜细胞增殖和凋亡的影响及其机制研究

The effect and mechanism of microRNA-448 on the proliferation and apoptosis of rheumatoid arthritis synovial cells

目的:探讨microRNA-448(miR-448)对类风湿关节炎滑膜细胞增殖和凋亡的影响及其机制。方法:类风湿关节炎滑膜细胞MH7A分成Control组、Anti-NC组(转染inhibitor control)、Anti-miR-448组(转染miR-448 inhibitor)、Anti-miR-448+si-NC组(共转染miR-448 inhibitor、siRNA control)、Anti-miR-448+si-程序性细胞死亡因子5(PDCD5)组(共转染miR-448 inhibitor、PDCD5 siRNA)。CCK-8检测细胞增殖,PI单染法检测细胞周期,Annexin V-FITC/PI双染法检测细胞凋亡,蛋白质印迹法检测C-Caspase-3、cyclin D1、p21蛋白表达。荧光素酶报告系统鉴定miR-448和PDCD5的靶向关系。结果:与Control组和Anti-NC组比较,Anti-miR-448组细胞存活率降低[(100.00±9.52)%、(99.71±10.36)%vs(52.62±4.47)%,P<0.05],凋亡率升高[(4.32±0.35)%、(4.18±0.62)%vs(12.51±1.32)%,P<0.05],G0/G1期比例升高[(48.51±3.26)%、(50.80±6.20)%vs(63.81±5.41)%,P<0.05],细胞中C-Caspase-3、p21蛋白表达水平升高,cyclin D1蛋白表达水平下降。与Anti-miR-448+si-NC组比较,Anti-miR-448+si-PDCD5组类风湿关节炎滑膜细胞存活率升高[(100.00±10.35)%vs(147.84±16.50)%,P<0.05],细胞G_(0)/G_(1)期比例降低[(64.08±6.10)%vs(53.11±4.75)%,P<0.05],细胞凋亡率降低[(13.05±1.16)%vs(6.92±0.35)%,P<0.05],细胞中C-Caspase-3、p21、PDCD5蛋白表达水平降低,cyclin D1蛋白表达水平升高。结论:下调的miR-448通过靶向上调PDCD5表达抑制类风湿关节炎滑膜细胞增殖,阻滞细胞周期,诱导细胞凋亡。

Objective:To investigate the effects of microRNA-448(miR-448)on the proliferation and apoptosis of synovial cells in rheumatoid arthritis and its mechanism.Methods:Rheumatoid arthritis synovial cells MH7 A were divided into Control group,Anti-NC group(transfected with inhibitor control),Anti-miR-448 group(transfected with miR-448 inhibitor),Anti-miR-448+si-NC group(co-transfected with miR-448 inhibitor,siRNA control),Anti-miR-448+si-PDCD5 group(co-transfected with miR-448 inhibitor,PDCD5 siRNA).Cell proliferation was detected by CCK-8,cell cycle was detected by PI single staining,cell apoptosis was detected by Annexin V-FITC/PI double staining,and protein expressions of C-Caspase-3,cyclin D1 and p21 were detected by Western blot.The luciferase reporting system was used to identify the targeting relationship between miR-448 and PDCD5.Results:Compared with Control group and Anti-NC group,Anti-miR-448 group cell survival rate decreased[(100.00±9.52)%,(99.71±10.36)%vs(52.62±4.47)%,P<0.05],apoptosis rate increased[(4.32±0.35)%,(4.18±0.62)%vs(12.51±1.32)%,P<0.05],G;/G;phase ratio increased[(48.51±3.26)%,(50.80±6.20)%vs(63.81±5.41)%,P<0.05],and C-Caspase-3 and p21 protein increased,and the expression level of cyclin D1 protein decreased.Compared with the Anti-miR-448+si-NC group,the survival rate of rheumatoid arthritis synovial cells in the Anti-miR-448+si-PDCD5 group was increased[(100.00±10.35)%vs(147.84±16.50)%,P<0.05],cell G_(0)/G_(1) phase ratio decreased[(64.08±6.10)%vs(53.11±4.75)%,P<0.05],cell apoptosis rate decreased[(13.05±1.16)%vs(6.92±0.35)%,P<0.05],the expression levels of C-Caspase-3,p21 and PDCD5 protein decreased,and the expression level of cyclin D1 protein increased.Down-regulation of miR-448 targeted up-regulation of PDCD5 expression.Conclusion:Down-regulation of miR-448 inhibits the proliferation of rheumatoid arthritis synovial cells,blocks the cell cycle and induces apoptosis by targeting the up-regulation of PDCD5 expression.