摘要
目的探讨人骨髓间充质干细胞(hBMMSCs)来源外泌体miRNA-320a调控CXC趋化因子配体9(CXCL9)表达在类风湿关节炎(RA)中的作用及机制。方法分离并鉴定h BMMSCs来源外泌体。收集于医院接受关节置换术的RA患者关节滑膜组织,制备类风湿关节炎滑膜成纤维细胞(RASFs)。q RT-PCR法检测RASFs和hBMMSCs来源外泌体中miRNA-320a和CXCL9表达。双荧光素酶报告基因验证miRNA-320a与CXCL9靶向关系。分别采用CCK-8法和Transwell实验,检测BMMSCs来源外泌体miRNA-320a通过下调CXCL9对RASFs增殖和迁移的影响。结果RASFs中miRNA-320a呈异常低表达,与hBMMSCs来源外泌体共培养后,miRNA-320a表达显著上调(P<0.05);miRNA-320a+CXCL9 WT组荧光强度显著低于miRNA-NC+CXCL9 WT组(P<0.05),而miRNA-320a+CXCL9 MUT组和miRNA-NC+CXCL9 MUT组荧光强度比较,差异无统计学意义(P>0.05);与miR-NC+oe-NC组比较,miR-320a mimic+oe-NC组miR-320a表达显著增加,CXCL9 mRNA表达显著降低,RASFs增殖和迁移能力显著下降(P<0.05);与miR-320a mimic+oe-NC组比较,miR-320a mimic+oe-CXCL9组CXCL9 mRNA表达显著增加,RASFs增殖和迁移能力显著上升(P<0.05)。结论h BMMSCs来源外泌体miRNA-320a可通过靶向下调CXCL9水平,抑制RASFs增殖和迁移,miR-320a有可能成为RA诊断标志物和潜在治疗靶点。
This study was designed to investigate the role and mechanism of exosomal miRNA-320 a derived from human mesenchymal stem cells(hBMMSCs)in the development and progression of rheumatoid arthritis(RA)via regulating CXC chemokine ligand 9(CXCL9)expression.The exosomes derived from mesenchymal stem cells were isolated and identified.At the same time,RA synovial fibroblasts(RASFs)were prepared from synovial tissue of RA patients who underwent joint replacement in our hospital.During the experiment,qRT-PCR was used to detect the expression of miRNA-320 a and CXCL9 in exosomes derived from RASFs or hBMMSCs.Dual luciferase reporter gene was used to verify the targeting relationship between miRNA-320 a and CXCL9.And CCK-8 method and Transwell experiment were used to detect the effect of mi RN A-320 a derived from BM MS Cs on the proliferation and migration of RASFs by down-regulating CXCL9.Data showed that the expression of miRNA-320 a in RASFs was abnormally low,but it was significantly up-regulated after co-culture with exosomes derived from hBMMSCs(P<0.05).After comparing,the fluorescence intensity of miRNA-320 a+CXCL9 WT group was significantly lower than that of miRNA-NC+CXCL9 WT group(P<0.05),while fluorescence intensity between miRNA-320 a+CXCL9 MUT group and miRNA-NC+CXCL9 MUT group were not statistically different(P>0.05).Compared with miR-NC+oe-NC group,the miR-320 a expression in miR-320 a mimic+oe-NC group was significantly increased,CXCL9 mRNA expression was significantly decreased,and the proliferation and migration ability of RASFs was significantly decreased(P<0.05).In addition,when compared with miR-320 a mimic+oe-NC group,the mRNA expression of CXCL9 in miR-320 a mimic+oe-CXCL9 group increased significantly,and the proliferation and migration ability of RASFs increased significantly(P<0.05).In conclusion,hBMMSCs-derived exosomes miRNA-320 a can down-regulate CXCL9 levels and inhibit the proliferation and migration of RASFs,indicating that miR-320 a may become a diagnostic marker and potential therapeutic target for RA.
作者
程明
吴杨玲
唐玉琦
高灿
肖云燚
刘羽
代浩
凌俐
CHENG Ming;WU Yangling;TANG Yuqi;GAO Can;XIAO Yunyi;LIU Yu;DAI Hao;LING Li(Department of Orthopedics,Sichuan Provincial People's Hospital,Chengdu 610072,China;Department of Rehabilitation,Sichuan Provincial People’s Hospital Jinniu Hospital,Chengdu 610036,China;Department of Psychosomatic Medicin,Sichuan Provincial People's Hospital,Chengdu 610072,China)
出处
《免疫学杂志》
CAS
CSCD
北大核心
2021年第12期1099-1104,共6页
Immunological Journal
基金
四川省科技计划(2020YFS0416)。
