Abstract Circular RNA sterile alpha motif domain containing 4A (circSAMD4A) was found to be differentially expressed in osteosarcoma and contributed to the tumorigenesis of osteosarcoma. However, the role of circSAMD4A in doxorubicin (DXR) resistance of osteosarcoma is yet to be elucidated. Levels of circSAMD4A, microRNA (miR)-218-5p and Krüppel-like factor 8 (KLF8) were detected using quantitative reverse transcription-polymerase chain reaction. Western blot was applied to detect the protein levels of KLF8, cyclin D1 and p21. Cell viability, cell cycle, migration and invasion were analyzed using Cell Counting Kit-8 assay, flow cytometry and transwell assay, respectively. The interaction between miR-218-5p and circSAMD4A or KLF8 was verified using dual-luciferase reporter assay or RNA immunoprecipitation assay. In vivo experiments were performed using murine xenograft models. CircSAMD4A and KLF8 were elevated in osteosarcoma, and knockdown of circSAMD4A or KLF8 sensitized osteosarcoma cells to DXR by mediating resistant cell viability, migration and invasion inhibition, and cell cycle arrest in vitro. miR-218-5p was decreased in osteosarcoma, and miR-218-5p inhibition enhanced DXR resistance. Besides, miR-218-5p was found to bind to circSAMD4A or KLF8, and subsequent rescue experiments indicated that miR-218-5p inhibition reversed the inhibitory effects of circSAMD4A silencing on DXR resistance, and silencing miR-218-5p enhanced DXR resistance by targeting KLF8 in osteosarcoma cells. Moreover, circSAMD4A could indirectly regulate KLF8 via miR-218-5p. Additionally, circSAMD4A knockdown enhanced the cytotoxicity of DXR in osteosarcoma in vivo via regulating miR-218-5p and KLF8. In all, circSAMD4A enhanced cell DXR resistance in osteosarcoma by regulating the miR-218-5p/KLF8 axis, suggesting a novel therapeutic target for therapy-resistant osteosarcoma.

中文翻译：

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CircSAMD4A 通过调节 miR-218-5p/KLF8 轴促进骨肉瘤中的细胞多柔比星抗性

摘要 含有 4A 的环状 RNA 无菌 α 基序结构域（circSAMD4A）被发现在骨肉瘤中差异表达，并有助于骨肉瘤的肿瘤发生。然而，circSAMD4A在骨肉瘤的多柔比星（DXR）抗性中的作用尚未阐明。使用定量逆转录聚合酶链反应检测 circSAMD4A、microRNA (miR)-218-5p 和 Krüppel 样因子 8 (KLF8) 的水平。应用蛋白质印迹法检测 KLF8、细胞周期蛋白 D1 和 p21 的蛋白质水平。分别使用 Cell Counting Kit-8 测定、流式细胞术和 Transwell 测定分析细胞活力、细胞周期、迁移和侵袭。使用双荧光素酶报告基因测定或 RNA 免疫沉淀测定验证了 miR-218-5p 与 circSAMD4A 或 KLF8 之间的相互作用。使用鼠异种移植模型进行体内实验。circSAMD4A 和 KLF8 在骨肉瘤中升高，敲除 circSAMD4A 或 KLF8 通过介导抗性细胞活力、迁移和侵袭抑制以及体外细胞周期停滞使骨肉瘤细胞对 DXR 敏感。骨肉瘤中 miR-218-5p 降低，抑制 miR-218-5p 增强 DXR 抗性。此外，发现miR-218-5p与circSAMD4A或KLF8结合，随后的拯救实验表明miR-218-5p抑制逆转了circSAMD4A沉默对DXR抗性的抑制作用，沉默miR-218-5p增强了DXR抗性靶向骨肉瘤细胞中的 KLF8。此外，circSAMD4A 可以通过 miR-218-5p 间接调节 KLF8。此外，circSAMD4A 敲低通过调节 miR-218-5p 和 KLF8 增强了 DXR 在体内 骨肉瘤中的细胞毒性。总之，circSAMD4A 通过调节 miR-218-5p/KLF8 轴增强了骨肉瘤中的细胞 DXR 抗性，这表明了抗治疗骨肉瘤的新治疗靶点。