Doxorubicin (DOX) is a widely used anticancer drug, but its cardiotoxicity largely limits its clinical utilization. Circular RNA spindle and kinetochore-associated protein 3 (circ-SKA3) were found to be differentially expressed in heart failure patients. In this study, we investigated the role and mechanism of circ-SKA3 in DOX-induced cardiotoxicity.

The quantitative real-time polymerase chain reaction and western blot assays were applied to measure the expression of circ-SKA3, microRNA (miR) -1303, and toll-like receptor 4 (TLR4). The viability and apoptosis of AC16 cells were analyzed using cell counting kit-8, flow cytometry, and western blot assays. The interaction between miR-1303 and circ-SKA3 or TLR4 was verified using dual-luciferase reporter and RNA immunoprecipitation assays. Exosomes were collected from culture media by the use of commercial kits and then qualified by transmission electron microscopy.

The expression of circ-SKA3 and TLR4 was increased, whereas miR-1303 expression was decreased in DOX-treated AC16 cells. DOX treatment promoted cell apoptosis and inhibited cell viability in AC16 cells in vitro, which was partially reversed by circ-SKA3 knockdown, TLR4 silencing, or miR-1303 overexpression. Mechanistically, circ-SKA3 served as a sponge for miR-1303 to upregulate TLR4, which was confirmed to be a target of miR-1303. Additionally, circ-SKA3 contributed to DOX-induced cardiotoxicity through the miR-1303/TLR4 axis. Further studies suggested that circ-SKA3 was overexpressed in exosomes extracted from DOX-mediated AC16 cells, which could be internalized by surrounding untreated AC16 cells.

Circ-SKA3 enhanced DOX-induced toxicity in AC16 cells through the miR-1303/TLR4 axis. Extracellular circ-SKA3 was packaged into exosomes, and exosomal circ-SKA3 could function as a mediator in intercellular communication between AC16 cells.

多柔比星（DOX）是一种广泛使用的抗癌药物，但其心脏毒性在很大程度上限制了其临床应用。发现环状 RNA 纺锤体和动粒相关蛋白 3（circ-SKA3）在心力衰竭患者中差异表达。在这项研究中，我们研究了 circ-SKA3 在 DOX 诱导的心脏毒性中的作用和机制。

应用定量实时聚合酶链反应和蛋白质印迹分析来测量 circ-SKA3、microRNA (miR) -1303 和 toll 样受体 4 (TLR4) 的表达。使用细胞计数试剂盒-8、流式细胞术和蛋白质印迹法分析 AC16 细胞的活力和凋亡。使用双荧光素酶报告基因和 RNA 免疫沉淀测定验证了 miR-1303 和 circ-SKA3 或 TLR4 之间的相互作用。通过使用商业试剂盒从培养基中收集外泌体，然后通过透射电子显微镜进行鉴定。

在 DOX 处理的 AC16 细胞中，circ-SKA3 和 TLR4 的表达增加，而 miR-1303 的表达降低。DOX 处理在体外促进 AC16 细胞的细胞凋亡并抑制细胞活力，这被 circ-SKA3 敲低、TLR4 沉默或 miR-1303 过表达部分逆转。从机制上讲，circ-SKA3 作为 miR-1303 的海绵来上调 TLR4，这被证实是 miR-1303 的靶标。此外，circ-SKA3 通过 miR-1303/TLR4 轴促进 DOX 诱导的心脏毒性。进一步的研究表明，circ-SKA3 在从 DOX 介导的 AC16 细胞中提取的外泌体中过度表达，这些外泌体可以被周围未处理的 AC16 细胞内化。

Circ-SKA3 通过 miR-1303/TLR4 轴增强 AC16 细胞中 DOX 诱导的毒性。细胞外 circ-SKA3 被包装成外泌体，外泌体 circ-SKA3 可以作为 AC16 细胞间细胞间通讯的介质。