lncRNA CASC2通过调控miR-218-5p表达对类风湿关节炎滑膜成纤维细胞炎症反应及迁移与侵袭的影响

Effects of lncRNA CASC2 on the inflammation,migration and invasion of rheumatoid arthritis fibroblasts by regulating the expression of miR-218-5p

目的探讨长链非编码RNA肿瘤易感候选基因2(CASC2)对类风湿关节炎(RA)滑膜成纤维细胞炎症反应、迁移及侵袭的影响及其机制。方法收集45例RA患者滑膜组织和18例健康对照者滑膜组织,采用qRT-PCR检测滑膜组织中CASC2和miR-218-5p的表达水平。ENCORI在线软件预测和双荧光素酶报告实验分析CASC2和miR-218-5p的靶向相互作用。将CASC2过表达质粒及miR-218-5p mimic转染至人RA滑膜成纤维细胞系MH7A中,qRT-PCR检测细胞中CASC2和miR-218-5p表达水平;CCK-8检测细胞的增殖水平;ELISA检测细胞上清液中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)水平;Transwell检测细胞迁移和侵袭能力;Western blot检测基质金属酶(MMP)-2和MMP-9蛋白表达水平。结果RA患者滑膜组织中CASC2表达水平较健康对照者滑膜组织减少(P<0.05),而miR-218-5p表达水平则增加(P<0.05)。双荧光素酶报告实验结果显示,CASC2与miR-218-5p之间存在靶向调控关系。过表达CASC2可抑制RA滑膜成纤维细胞MH7A的增殖活性,降低细胞上清液中TNF-α、IL-1β、IL-6水平,减弱细胞迁移及侵袭能力,下调细胞中miR-218-5p及MMP-2和MMP-9蛋白表达水平。然而,转染miR-218-5p mimic可逆转CASC2过表达对MH7A细胞增殖、迁移与侵袭及炎症反应的抑制作用。结论CASC2在RA患者滑膜组织中低表达,其过表达可抑制RA滑膜成纤维细胞炎症反应及迁移与侵袭能力,其作用机制可能与靶向负调节miR-218-5p表达相关。

Objective To investigate the effects effect of lncRNA CASC2 on inflammation,migration and invasion of rheumatoid arthritis(RA)synovial fibroblasts by regulating the expression of miR-218-5 p and its mechanism.Methods 45 cases of RA synovial tissue and 18 cases of normal synovial tissue were collected,and qRT-PCR was used to detect the expression of CASC2 and miR-218-5 p in the synovial tissue.ENCORI prediction and dual luciferase reporting experiments analyzed the targeting between CASC2 and miR-218-5 p.RA synovial fibroblast MH7 A were transfected with pcDNA3.1-CASC2 and miR-218-5 p mimic,the expression of CASC2 and miR-218-5 p was detected by qRT-PCR;CCK-8 was used to detect cell proliferation;ELISA was used to detect the levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)in the supernatant of cells;Transwell was used to detect migration and invasion of cells;Western blot was used to detect expression of matrix metalloenzymes MMP-2 and MMP-9.Results Compared with normal synovial tissue,RA synovial tissue showed significantly down-regulated expressions of CASC2(P<0.05)and up-regulated expressions of miR-1236-3 p(P<0.05).The dual luciferase reporter showed that CASC2 was target genes of miR-218-5 p.Overexpression of CASC2 significantly suppressed cell proliferation,reduced the levels of TNF-α,IL-1βand IL-6 in the supernatant and migration and invasion,lowered the levels of miR-218-5 p and protein expression of MMP-2 and MMP-9 in RA synovial fibroblast MH7 A.However,transfecting miR-218-5 p mimic could reverse the situation.Conclusion RA synovial tissue showed significantly down-regulated expressions of CASC2,overexpression of CASC2 suppressed inflammation,migration and invasion of RA fibroblasts by regulating the expression of miR-218-5 p.