长链非编码RNA核受体亚家族2 F组成员2反义RNA 1靶向微小RNA-588调控类风湿关节炎滑膜成纤维细胞增殖、迁移侵袭的分子机制

The molecular mechanism of long noncoding RNA NR2F2-AS1 targeting microRNA-588 to regulate the proliferation,migration and invasion of rheumatoid arthritis synovial fibroblasts

目的探讨长链非编码RNA核受体亚家族2 F组成员2反义RNA 1(lncRNA NR2F2-AS1)对类风湿关节炎滑膜成纤维细胞(RASF)增殖、迁移侵袭的影响及分子机制。方法选取郑州大学第一附属医院35例RA患者滑膜组织及正常滑膜组织,实时荧光定量聚合酶链反应(RT-qPCR)检测NR2F2-AS1和微小RNA(miR)-588的表达水平;将RASF分为si-NR2F2-AS1组、si-NC组、miR-588组、miR-NC组、si-NR2F2-AS1+anti-miR-588组、si-NR2F2-AS1+anti-miR-NC组;细胞计数试剂盒(CCK-8)检测细胞活性;克隆形成实验检测细胞克隆形成数;划痕实验检测细胞划痕愈合率;Transwell检测细胞侵袭数;蛋白质印迹法检测蛋白表达;双荧光素酶报告实验检测NR2F2-AS1和miR-588的靶向关系。用SPSS 20.0软件进行统计学分析。结果 RA患者滑膜组织中NR2F2-AS1表达水平高于正常滑膜组织(3.50±0.23比1.00±0.06,t=62.223,P<0.05),而miR-588表达水平低于正常滑膜组织(0.29±0.04比1.00±0.09,t=42.649,P<0.05)。si-NR2F2-AS1组细胞活性低于si-NC组(0.55±0.04比1.03±0.08,t=16.100,P<0.05),细胞克隆形成数低于si-NC组[(47.58±5.03)个比(103.93±10.24)个,t=14.818,P<0.05]、侵袭细胞数[(55.31±5.28)个比(123.37±10.09)个,t=17.929,P<0.05]低于si-NC组,划痕愈合率低于si-NC组[(23.21±2.47)%比(64.19±5.55)%,t=20.238,P<0.05],E钙黏蛋白(E-cadherin)表达水平高于si-NC组,N钙黏蛋白(N-cadherin)表达水平低于si-NC组(P<0.05)。过表达miR-588与其作用相同。NR2F2-AS1靶向调控miR-588;下调miR-588表达逆转抑制NR2F2-AS1表达对RASF增殖、迁移侵袭的作用。结论抑制lncRNA NR2F2-AS1表达可通过靶向调控miR-588抑制RA滑膜成纤维细胞的增殖、迁移侵袭。

Objective To explore the effect of long non-coding RNA nuclear receptor subfamily 2 F group member 2 antisense RNA 1(lncRNA NR2F2-AS1)on the proliferation,migration and invasion of rheumatoid arthritis synovial fibroblasts(RASF)and its molecular mechanism.Methods Select 35 patients with rheumatoid arthritis synovium tissue from the Department of Orthopedics,First Affiliated Hospital of Zhengzhou University,real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)to detect the expression levels of NR2F2-AS1 and microRNA(miR)-588;RASF was divided into si-NR2F2-AS1 group,si-NC group,miR-588 group,miR-NC group,si-NR2F2-AS1+anti-miR-588 group,si-NR2F2-AS1+anti-miR-NC group;cell counting kit-8(CCK-8)to detect cell viability;clone formation experiment to detect the number of cell clone formation;the scratch test was used to detect the healing rate of cell scratches;the number of cell invasion was detected by Transwell;the protein expression was detected by Western blotting;the dual luciferase reporter test was used to detect the targeting relationship between NR2F2-AS1 and miR-588.Statistical analysis was performed with SPSS 20.0 software.Results The expression level of NR2F2-AS1 in the synovial tissue of RA patients was higher than that of normal synovial tissue(3.50±0.23 vs.1.00±0.06,t=62.223,P<0.05),while the expression level of miR-588 was lower than that of normal synovial tissue(0.29±0.04 vs.1.00±0.09,t=42.649,P<0.05).The cell viability of the si-NR2F2-AS1 group was lower than that of the si-NC group(0.55±0.04 vs.1.03±0.08,t=16.100,P<0.05),and the number of cell clone formation was lower than that of the si-NC group[(47.58±5.03)vs.(103.93±10.24),t=14.818,P<0.05],the number of invasive cells[(55.31±5.28)vs.(123.37±10.09),t=17.929,P<0.05]was lower than the si-NC group,the scar healing rate was lower than that of the si-NC group[(23.21±2.47)%to(64.19±5.55)%,t=20.238,P<0.05],and the expression level of E-cadherin was higher than that of the si-NC group,the expression level of N-cadherin was lower than that of the si-NC group(P<0.05).Overexpression of miR-588 has the same effect.NR2F2-AS1 targets and regulates miR-588;down-regulation of miR-588 expression reversed the effect of inhibiting NR2F2-AS1 expression on the proliferation,migration and invasion of RASF.Conclusion Inhibiting the expression of lncRNA NR2F2-AS1 can inhibit the proliferation,migration and invasion of rheumatoid arthritis synovial fibroblasts through targeted regulation of miR-588.