环状RNA ADP核糖基化因子GTP酶激活蛋白调控类风湿关节炎滑膜成纤维细胞增殖及迁移机制  

circular RNA Arfgap with sh3 domain,ankyrin repeat and ph domain 1 regulates the proliferation and migration of rheumatoid arthritis synovial fibroblasts

作　　者：欧阳峰松[1,2] 向忠军 熊瑛[2] 王少林 丁乐[2] 曹彪 Ouyang Fengsong;Xiang Zhongjun;Xiong Ying;Wang Shaolin;Ding Le;Cao Biao(Hunan Provincial Key Laboratory of New Drug Preparation Research and Development,Changsha Medical University,Changsha 410219,China;College of Traditional Chinese Medicine,Changsha Medical University,Changsha 410219,China;Department of Orthopedics,the First Affiliated Hospital of Changsha Medical College,Changsha 410219,China)

机构地区：[1]长沙医学院新型药物制剂研发湖南省重点实验室,420219 [2]长沙医学院中医学院,410219 [3]长沙医学院附属第一医院骨科,410219

出　　处：《中华实验外科杂志》2022年第11期2105-2108,共4页Chinese Journal of Experimental Surgery

基　　金：湖南省普通高校中医类创新创业教育中心项目(湘教通[2020]301号79);长沙医学院中医学校级重点学科项目(长医研[2016]3号)。

摘　　要：目的探讨环状RNA(circRNA)ADP核糖基化因子GTP酶激活蛋白(ASAP1)调控类风湿关节炎(RA)滑膜成纤维细胞(SFs)增殖及迁移机制。方法提取RA及关节外伤患者SFs行后续实验。分别用Lipofectamine 3000将si-NC、si-circASAP1、miR-NC、miR-144-3p mimics、pcDNA-NC、pcDNA-circASAP1、抗miR-NC与si-circASAP1、抗miR-144-3p与si-circASAP1分别转染RA-SFs(记为si-NC组、si-circASAP1组、miR-NC组、miR-144-3p组、pcDNA-NC组、pcDNA-circASAP1组、抗miR-NC+si-circASAP1组、抗miR-144-3p+si-circASAP1组)。未转染RA-SFs记为对照组(NC组)。细胞计数试剂盒-8(CCK-8)检测细胞增殖;实时荧光定量聚合酶链式反应(qRT-PCR)检测circASAP1和微小核糖核酸144-3p(miR-144-3p)表达;蛋白质印迹法检测基质金属蛋白酶2(MMP-2)及基质金属蛋白酶9(MMP-9)蛋白表达;Transwell检测细胞迁移;双荧光素酶报告基因实验检测circASAP1靶向调控miR-144-3p表达。两组间比较采用t检验;多组间比较用单因素方差分析(两组间比较用SNK-q检验)。结果RA患者滑膜组织及SFs中circASAP1表达高于关节外伤患者滑膜组织及SFs(滑膜组织2.46±0.28比1.00±0.12,SFs中3.16±0.15比1.00±0.11),miR-144-3p低于关节外伤患者滑膜组织及SFs(滑膜组织0.43±0.11比1.00±0.15,SFs中0.38±0.03比1.00±0.08),差异有统计学意义(t=40.949、26.182,P<0.05)。si-circASAP1组RA-SFs的circASAP1、MMP-2、MMP-9表达、细胞活力和细胞迁移数均低于NC组和si-NC组(0.47±0.03比1.00±0.07、1.02±0.10,0.41±0.03比0.83±0.07、0.81±0.06,0.35±0.03比0.77±0.06、0.75±0.06,0.57±0.05比1.12±0.07、1.11±0.09,97.00±7.23比194.00±13.25、203.00±11.54),差异有统计学意义(F=166.272、161.234、187.111、194.126、258.352,P<0.05)。miR-144-3p组RA-SFs的miR-144-3p表达高于miR-NC组(2.09±0.15比1.00±0.09),MMP-2、MMP-9表达、细胞活力和细胞迁移数均低于miR-NC组(0.37±0.02比0.83±0.07、0.31±0.02比0.74±0.06、0.62±0.05比1.21±0.07、115.00±7.36比197.00±12.51),差异Objective To investigate the mechanism of circular RNA(circRNA)ADP ribosylation factor GTPase activator protein(Arfgap with sh3 domain,ankyrin repeat and ph domain 1,ASAP1)in the regulation of synovial fibroblasts’(SFs)proliferation and migration in rheumatoid arthritis(RA).Methods SFs from patients with RA and joint trauma were extracted for follow-up experiments.SFs from patients with RA and joint trauma were extracted for follow-up experiments.si-NC,si-circASAP1,miR-NC,miR-144-3p mimics,pcDNA-NC,pcDNA-circASAP1,anti-miR-NC and si-circASAP1,anti-miR-144-3p,si-circASAP1 were transfected into RA-SFs(marked as si-NC group,si-circASAP1 group,miR-NC group,miR-144-3p group,pcDNA-NC group,pcDNA-circASAP1 group,anti-miR-NC+si-circASAP1 group,anti-miR-144-3p+si-circASAP1 group).Untransfected RA-SFs were recorded as control group(NC group).Cell counting kit-8(CCK-8)was used to detect cell proliferation;Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect circASAP1 and micro ribonucleic acid-144-3p(miR-144-3p)expression;Western blotting detection of matrix metalloproteinase-2(MMP-2)and matrix metalloproteinase-9(MMP-9))protein expression;Transwell detected cell migration;dual luciferase reporter gene assay detected the targeted regulation of miR-144-3p expression by circASAP1.The t test was used for comparison between two groups;the one-way analysis of variance was used for comparison between multiple groups(SNK-q test was used for comparison between two groups).The circASAP1,MMP-2,MMP-9 expression,cell viability and cell migration number of RA-SFs in the si-circASAP1 group were lower than those in the NC and si-NC groups(0.47±0.03 to 1.00±0.07,1.02±0.10,0.41±0.03 to 0.83±0.07,0.81±0.06,0.35±0.03 to 0.77±0.06,0.75±0.06,0.57±0.05 to 1.12±0.07,1.11±0.09,97.0±7.23 vs.194.00±13.25,203.00±11.54),the difference was statistically significant(F=166.272,161.234,187.111,194.126,258.352,P<0.05).The expression of miR-144-3p in RA-SFs in the miR-144-3p group was higher than that in the miR-NC gr

关 键 词：类风湿关节炎 环状RNA ADP核糖基化因子GTP酶激活蛋白 微小RNA-144-3p 细胞增殖 细胞迁移 

分 类 号：R593.22[医药卫生—内科学]