Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China.
Spine (Phila Pa 1976). 2023 Dec 1;48(23):E401-E408. doi: 10.1097/BRS.0000000000004777. Epub 2023 Aug 9.
Experimental analysis of circular RNA in intervertebral disk degeneration (IDD).
This study aimed to explore the roles of hsa_circ_0001946 (circ-CDR1as) in mechanical stress-induced nucleus pulposus cell injury in IDD.
Mechanical stress is an important pathogenic factor for IDD. Excessive compression stress leads to nucleus pulposus (NP) cell apoptosis and extracellular matrix (ECM) degradation and accelerated IDD. Circ-CDR1as is associated with various degenerative conditions, but its role in IDD is not clear. Herein, we explored the roles and mechanisms of circ-CDR1as in IDD in vitro.
An in vitro model of IDD was constructed by treating NP cells with 1.0 MPa compression stress. Quantitative real-time polymerase chain reaction assay was used for detecting the expression of circ-CDR1as and miR-432-5p. Immunofluorescent analysis was performed for MMP13 detection. Western blot assay was performed for detecting apoptosis and ECM-related protein expression. Flow cytometry analysis was used for cell apoptosis analysis. The dual-luciferase reporter was used to analyze the interaction between miR-432-5p and circ-CDR1as or SOX9. Differences in means between groups were evaluated using the Student t test or one-way analysis of variance.
In compression-treated human NP cells, we found that circ-CDR1as was significantly downregulated. Functional experiments showed that circ-CDR1as overexpression reduced the compression-induced apoptosis and ECM degradation in NP cells. Further research indicated that circ-CDR1as could act as a molecular sponge for miR-432-5p, a miRNA that enhanced compression-induced damage of NP cells by inhibiting the expression of SOX9. The luciferase reporter experiments also showed that the mutual dialogue between circ-CDR1as and miR-432-5p regulated the expression of SOX9.
Circ-CDR1as binds to miR-432-5p and plays a protective role in mitigating compression-induced NP cell apoptosis and ECM degradation by targeting SOX9. Circ-CDR1as may provide a novel therapeutic target for the clinical management of IDD in the future.
椎间盘退变（IDD）中环状 RNA 的实验分析。
本研究旨在探讨 hsa_circ_0001946（circ-CDR1as）在机械应力诱导的 IDD 中对核髓核细胞损伤的作用。
机械应力是 IDD 的一个重要致病因素。过大的压缩应力导致核髓核（NP）细胞凋亡和细胞外基质（ECM）降解，加速 IDD。Circ-CDR1as 与各种退行性疾病有关，但在 IDD 中的作用尚不清楚。在此，我们在体外探讨了 circ-CDR1as 在 IDD 中的作用和机制。
通过用 1.0 MPa 压缩力处理 NP 细胞构建 IDD 的体外模型。定量实时聚合酶链反应检测 circ-CDR1as 和 miR-432-5p 的表达。免疫荧光分析用于检测 MMP13。通过 Western blot 检测分析凋亡和 ECM 相关蛋白的表达。通过流式细胞术分析检测细胞凋亡。双荧光素酶报告用于分析 miR-432-5p 与 circ-CDR1as 或 SOX9 之间的相互作用。使用 Student t 检验或单因素方差分析评估组间均值的差异。
在受压缩处理的人 NP 细胞中，我们发现 circ-CDR1as 表达明显下调。功能实验表明，circ-CDR1as 过表达可减少 NP 细胞的压缩诱导性凋亡和 ECM 降解。进一步的研究表明，circ-CDR1as 可以作为 miR-432-5p 的分子海绵，miR-432-5p 通过抑制 SOX9 的表达来增强 NP 细胞的压缩诱导损伤。荧光素酶报告实验也表明 circ-CDR1as 与 miR-432-5p 的相互作用调节了 SOX9 的表达。
Circ-CDR1as 与 miR-432-5p 结合，通过靶向 SOX9 发挥减轻压缩诱导的 NP 细胞凋亡和 ECM 降解的保护作用。Circ-CDR1as 可能为未来 IDD 的临床管理提供新的治疗靶点。
