circCBLB抑制类风湿性关节炎成纤维细胞样滑膜细胞增殖促进细胞凋亡并增加抗炎细胞因子水平  

Circular RNA Cbl proto-oncogene B(circCBLB)inhibits proliferation,promotes apoptosis,and increases anti-inflammatory cytokine levels of fibroblast-like synoviocytes in rheumatoid arthritis

作　　者：李舒 万磊[1] 刘健[1] 黄传兵[1] 陈莹莹 程静 胡赛赛 李方泽 朱子衡 LI Shu;WAN Lei;LIU Jian;HUANG Chuanbing;CHEN Yingying;CHENG Jing;HU Saisai;LI Fangze;ZHU Ziheng(Department of Rheumatism Immunity,First Affiliated Hospital of Anhui University of Traditional Chinese Medicine,Hefei 230031,China)

机构地区：[1]安徽中医药大学第一附属医院风湿科,安徽合肥230031

出　　处：《细胞与分子免疫学杂志》2024年第2期106-113,共8页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81973655,82274501);新安医学与中医药现代化研究所揭榜挂帅项目(2023CXMMTCM020);国家中医药管理局第七批全国老中医药专家学术经验继承项目(国中医药人教函[2022]76号);安徽省自然科学基金(2308085MH291);安徽省高校优秀青年人才支持计划项目(皖教秘人[2022]11号);新安医学教育部重点实验室(2020xayx04);安徽省高校自然科学研究项目(2023AH050814);安徽省高等学校质量工程项目省级教育教学改革研究项目(2022jyxm883);安徽省新时代育人质量工程项目(2022xscx103)。

摘　　要：目的 探讨环状RNA Cbl原癌基因B(circCBLB)/miR-486-5p功能轴对类风湿性关节炎成纤维细胞样滑膜细胞(RA-FLS)增殖、凋亡及炎症因子产生的影响。方法 人RA-FLS,用100μL的10 ng/mL肿瘤坏死因子α(TNF-α)刺激RA-FLS建立模型。采用双荧光素酶靶向验证circCBLB/miR-486-5p的结合关系。构建pcDNA3.1/siRNA-circCBLB、阴性对照(pcDNA3.1-NC/si-NC)和mimics-miR-486-5p,分别转染至RA-FLS。实验分为对照组、 TNF-α处理的RA-FLS、 pcDNA3.1-circCBLB组、 pcDNA3.1-NC组、 si-circCBLB组、 si-NC组、 pcDNA3.1-circCBLB联合miR-486-5p-mimics组。采用CCK-8法检测细胞活力,流式细胞术检测细胞周期与细胞凋亡,平板集落形成实验检测细胞平板集落形成能力,实时定量PCR检测各组circCBLB、 miR-486-5p表达水平,ELISA检测各组细胞上清液抗炎因子白细胞介素4(IL-4)、 IL-10,促炎因子IL-6、 TNF-α水平。结果 双荧光素酶报告基因结果示circCBLB与miR-486-5p的3′非翻译区(3′UTR)结合;与同一时间点模型组细胞相比,过表达组细胞活力降低,干扰组升高;与模型组相比,过表达组凋亡率升高、 S和G2期的比例升高、集落形成率降低、 miR-486-5p表达水平降低、 IL-4、 IL-10水平升高,IL-6、 TNF-α水平降低;干扰组凋亡率降低、 S和G2期的比例降低、集落形成率升高、 miR-486-5p表达水平升高、 TNF-α水平升高;pcDNA3.1-circCBLB联合miR-486-5p-mimics组在circCBLB过表达的基础上,在细胞活力、细胞凋亡率、细胞周期、集落形成能力、抗炎因子的提高、促炎因子的降低方面对circCBLB的效应产生了抵消与逆转。结论 circCBLB抑制RA-FLS活力、提高细胞凋亡率、延长细胞周期降低细胞集落能力、提高抗炎因子,降低促炎因子,miR-486-5p则与circCBLB调控能力相反,且对circCBLB的效应具有一定的抵消与逆转能力。Objective To explore the regulatory axis of circular RNA Cbl proto-oncogene B(circCBLB)/miR-486-5p on the proliferation,apoptosis,and inflammatory cytokines of fibroblast-like synoviocytes in rheumatoid arthritis(RA-FLS).Methods Human RA-FLS were stimulated with 100μL of 10 ng/mL of tumor necrosis factor-alpha(TNF-α)to establish the model.The binding relationship of circCBLB/miR-486-5p was validated by a dual-luciferase reporter gene assay.pcDNA3.1/siRNA-circCBLB,negative control(pcDNA3.1-NC/si-NC),and miR-486-5p-mimics were created and transfected into RA-FLS,respectively.The experiment was divided into seven groups:control,TNF-α-treated RA-FLS,pcDNA3.1-circCBLB,pcDNA3.1-NC,si-circCBLB,si-NC,and pcDNA3.1-circCBLB combined with miR-486-5p-mimics.Cell viability was assessed by a CCK-8 assay;cell cycle and apoptosis by flow cytometry;colony formation ability by a colony formation assay;and the expression levels of circCBLB and miR-486-5p by real-time quantitative PCR.The levels of interleukin 4(IL-4),IL-10,IL-6 and TNF-αwere measured by ELISA.Results The dual-luciferase reporter gene assay showed that circCBLB bound to the 3'untranslated region(3'UTR)of miR-486-5p.Compared with the model group at the same time point,the cell viability of the overexpression group was lower,while that of the interference group was higher.Compared with the model group,the overexpression group had a higher apoptosis rate,a higher proportion in S and G2 phases,a lower colony formation rate,a lower miR-486-5p expression level,higher IL-4 and IL-10 levels,and lower IL-6 and TNF-αlevels.The interference group had a lower apoptosis rate,a lower proportion in S and G2 phases,a higher colony formation rate,a higher miR-486-5p expression level,and a higher TNF-αlevel.The pcDNA3.1-circCBLB combined with miR-486-5p-mimics group reversed the effects of circCBLB on cell viability,apoptosis rate,cell cycle,colony formation ability,antinflammatory cytokines,and proinflammatory cytokines.Conclusion circCBLB inhibits the viability of RA-FLS,incr

关 键 词：环状RNACbl原癌基因B(circCBLB) miR-486-5p 细胞增殖 细胞凋亡 炎症因子 

分 类 号：R364.5[医药卫生—病理学] R593.22[医药卫生—基础医学] R684.3R392.12