Wei Wei, Ji Liefeng, Duan Wanli, Zhu Jiang
Department of orthopedics, Shaoxing Shangyu People's Hospital, No. 517, Shimin Avenue, Baiguan Street, Shangyu District, Shaoxing, Zhejiang Province, 312300, China.
Open Life Sci. 2020 Nov 24;15(1):848-859. doi: 10.1515/biol-2020-0079. eCollection 2020.
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. 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 . 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 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.
环状RNA含无菌α基序结构域4A（circSAMD4A）在骨肉瘤中存在差异表达，并促进了骨肉瘤的肿瘤发生。然而，circSAMD4A在骨肉瘤对多柔比星（DXR）耐药中的作用尚待阐明。采用定量逆转录-聚合酶链反应检测circSAMD4A、微小RNA（miR）-218-5p和Krüppel样因子8（KLF8）的水平。应用蛋白质免疫印迹法检测KLF8、细胞周期蛋白D1和p21的蛋白水平。分别使用细胞计数试剂盒-8检测法、流式细胞术和Transwell检测法分析细胞活力、细胞周期、迁移和侵袭情况。使用双荧光素酶报告基因检测法或RNA免疫沉淀检测法验证miR-218-5p与circSAMD4A或KLF8之间的相互作用。实验采用小鼠异种移植模型进行。circSAMD4A和KLF8在骨肉瘤中升高，敲低circSAMD4A或KLF8可通过介导耐药细胞活力、抑制迁移和侵袭以及使细胞周期停滞，使骨肉瘤细胞对DXR敏感。miR-218-5p在骨肉瘤中表达降低，抑制miR-218-5p可增强DXR耐药性。此外，发现miR-218-5p与circSAMD4A或KLF8结合，随后的挽救实验表明抑制miR-218-5p可逆转circSAMD4A沉默对DXR耐药性的抑制作用，并且在骨肉瘤细胞中沉默miR-218-5p通过靶向KLF8增强DXR耐药性。此外，circSAMD4A可通过miR-218-5p间接调节KLF8。另外，敲低circSAMD4A通过调节miR-218-5p和KLF8增强了DXR对骨肉瘤的细胞毒性。总之，circSAMD4A通过调节miR-218-5p/KLF8轴增强了骨肉瘤细胞对DXR的耐药性，提示其可能是治疗耐药性骨肉瘤的一个新的治疗靶点。
