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.
Highlights
Osteosarcoma is the most frequent primary solid malignancy of bone, with a higher incidence in children and adolescents [1]
Osteosarcoma tissues were divided into the DXR-resistant group and the DXR-sensitive group depending on the sensitivity of osteosarcoma patients to DXR, and we found that circSAMD4A (Figure 1c) and Krüppel-like factor 8 (KLF8) (Figure 1k and o) were notably higher, while miR-218-5p (Figure 1g) was lower in the treatment-resistant group than those in the treatment-responsive group
We found that the number of HOS/DXR and U2OS/DXR cells in the S phase was decreased upon circSAMD4A silencing, while cells in the G0/G1 phase were accumulated, indicating cell cycle arrest (Figure 2d and e); the downregulation of cyclin D1 levels and upregulation of p21 levels induced by circSAMD4A knockdown in HOS/DXR and U2OS/DXR cells further suggested the cell cycle arrest (Figure 2f and g)
Summary
Osteosarcoma is the most frequent primary solid malignancy of bone, with a higher incidence in children and adolescents [1]. Recent studies have suggested that non-coding RNAs (ncRNAs) and ncRNA-regulatory processes are involved in drug resistance in multiple types of cancers [4]. It has been documented that ncRNAs function as underlying players in multiple cellular processes, including cell cycle, differentiation, proliferation, metastasis, angiogenesis and oxidative stress [6,7,8]. Circular RNAs (circRNAs) are a new class of highly conserved ncRNAs forming a covalently closed continuous loop. Emerging evidence has identified the association between circRNAs and drug resistance in osteosarcoma [9,10]. Circular RNA sterile alpha motif domain containing 4A (circSAMD4A) is a newly identified circRNA, and Yanbin et al found that circSAMD4A enhanced cell proliferation and the
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