外泌体 circ-BRWD1 通过调节 miR-1277/TRAF6 轴促进骨关节炎的发展。
Exosomal circ-BRWD1 contributes to osteoarthritis development through the modulation of miR-1277/TRAF6 axis.
机构信息
Department of Orthopaedics, Second Hospital of Shanxi Medical University, No.2 Hospital, Shanxi Medical University, 382 Wuyi Road, Xinghualing District, Taiyuan City, Shanxi Province, China.
Department of Orthopaedics, Shanxi Medical University, West Hospital, Second Hospital, Shanxi Medical University, 379 Yingzhi Street, Wanbai District, Taiyuan City, Shanxi Province, China.
出版信息
Arthritis Res Ther. 2021 Jun 3;23(1):159. doi: 10.1186/s13075-021-02541-8.
BACKGROUND
Circular RNAs (circRNAs) can act as vital players in osteoarthritis (OA). However, the roles of circRNAs in OA remain obscure. Herein, we explored the roles of exosomal circRNA bromodomain and WD repeat domain containing 1(circ-BRWD1) in OA pathology.
METHODS
In vitro model of OA was constructed by treating CHON-001 cells with interleukin-1β (IL-1β). Quantitative real-time polymerase chain reaction (qRT-PCR) assay was used for circ-BRWD1, BRWD, miR-1277, and TNF receptor-associated factor 6 (TRAF6) levels. RNase R assay was conducted for the feature of circ-BRWD1. Transmission electron microscopy (TEM) was employed to analyze the morphology of exosomes. Western blot assay was performed for protein levels. Cell Counting Kit-8 (CCK-8) assay, flow cytometry analysis, and 5-Ethynyl-2'-deoxyuridine (EDU) assay were adopted for cell viability, apoptosis, and proliferation, respectively. Enzyme-linked immunosorbent assay (ELISA) was carried out for the concentrations of interleukin-6 (IL-6) and interleukin-8 (IL-8). Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to analyze the interaction between miR-1277 and circ-BRWD1 or TRAF6.
RESULTS
Circ-BRWD1 was increased in OA cartilage tissues, IL-1β-treated CHON-001 cells, and the exosomes derived from IL-1β-treated CHON-001 cells. Exosome treatment elevated circ-BRWD1 level, while exosome blocker reduced circ-BRWD1 level in IL-1β-treated CHON-001 cells. Silencing of circ-BRWD1 promoted cell viability and proliferation and repressed apoptosis, inflammation, and extracellular matrix (ECM) degradation in IL-1β-stimulated CHON-001 cells. For mechanism analysis, circ-BRWD1 could serve as the sponge for miR-1277 to positively regulate TRAF6 expression. Moreover, miR-1277 inhibition ameliorated the effects of circ-BRWD1 knockdown on IL-1β-mediated CHON-001 cell damage. Additionally, miR-1277 overexpression relieved IL-1β-induced CHON-001 cell injury, while TRAF6 elevation restored the impact.
CONCLUSION
Exosomal circ-BRWD1 promoted IL-1β-induced CHON-001 cell progression by regulating miR-1277/TRAF6 axis.
背景
环状 RNA（circRNA）可以作为骨关节炎（OA）的重要参与者。然而，circRNA 在 OA 中的作用仍不清楚。本研究旨在探讨外泌体 circBRWD1 在 OA 病理中的作用。
方法
用白细胞介素-1β（IL-1β）处理 CHON-001 细胞构建 OA 的体外模型。采用实时定量聚合酶链反应（qRT-PCR）检测 circBRWD1、BRWD、miR-1277 和肿瘤坏死因子受体相关因子 6（TRAF6）水平。用核糖核酸酶 R 试验检测 circBRWD1 的特征。采用透射电子显微镜（TEM）分析外泌体的形态。采用 Western blot 检测蛋白水平。用细胞计数试剂盒-8（CCK-8）检测细胞活力，用流式细胞术分析和 5-乙炔基-2'-脱氧尿苷（EDU）检测细胞凋亡和增殖。用酶联免疫吸附试验（ELISA）检测白细胞介素-6（IL-6）和白细胞介素-8（IL-8）的浓度。用双荧光素酶报告和 RNA 免疫沉淀（RIP）试验分析 miR-1277 与 circBRWD1 或 TRAF6 的相互作用。
结果
OA 软骨组织、IL-1β 处理的 CHON-001 细胞和 IL-1β 处理的 CHON-001 细胞来源的外泌体中 circBRWD1 水平升高。外泌体处理可升高 circBRWD1 水平，而外泌体阻断剂可降低 IL-1β 处理的 CHON-001 细胞中的 circBRWD1 水平。沉默 circBRWD1 可促进 IL-1β 刺激的 CHON-001 细胞活力和增殖，并抑制细胞凋亡、炎症和细胞外基质（ECM）降解。机制分析表明，circBRWD1 可作为 miR-1277 的海绵，正向调节 TRAF6 表达。此外，抑制 miR-1277 可改善 circBRWD1 敲低对 IL-1β 介导的 CHON-001 细胞损伤的作用。此外，miR-1277 过表达可缓解 IL-1β 诱导的 CHON-001 细胞损伤，而 TRAF6 升高则可恢复其影响。
结论
外泌体 circBRWD1 通过调节 miR-1277/TRAF6 轴促进 IL-1β 诱导的 CHON-001 细胞进展。
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