Increasing evidence reveals that circular RNAs (circRNAs) serve as oncogenes or tumor suppressors in the development of various tumors including bladder cancer (BCa). In this study, we explored the function and mechanism of circ_0030586 (also named circABCC4, ATP binding cassette subfamily C member 4) in BCa. The expression of circ_0030586 was significantly decreased in BCa tissues and cells, as suggested by RT-qPCR. The circular characteristics of circ_0030586 were verified by agarose gel electrophoresis and RNase R treatment. Colony formation, 5-Ethynyl-2′-deoxyuridine and sphere formation assays revealed that overexpression of circ_0030586 suppressed BCa cell proliferation and stemness in vitro. According to xenograft experiment, circ_0030586 overexpression suppressed tumor growth in vivo. Mechanistically, RNA pulldown and luciferase reporter assays were carried out to explore the interaction between genes. Circ_0030586 served as a competing endogenous RNA (ceRNA) for hsa-miR-665 to upregulate the expression of nuclear receptor subfamily 4 group A member 3 (NR4A3) which is a downstream target gene of miR-665 in BCa. MiR-665 exhibited high expression in BCa tissues and cells while NR4A3 expression was downregulated in BCa. MiR-665 overexpression or NR4A3 silencing reversed the suppressive effect of circ_0030586 overexpression on BCa cell proliferation and stemness. Moreover, western blot analysis revealed that circ_0030586 inactivated the extracellular signal-regulated kinase (ERK) pathway by upregulating NR4A3 expression. In conclusion, circ_0030586 inhibits BCa cell proliferation and stemness by serving as a ceRNA for miR-665 to upregulate NR4A3 expression and thus inactivate the ERK signaling.

越来越多的证据表明，环状 RNA (circRNA) 在包括膀胱癌 (BCa) 在内的各种肿瘤的发展中充当癌基因或肿瘤抑制因子。在这项研究中，我们探索了 circ_0030586（也称为 circABCC4，ATP 结合盒亚科 C 成员 4）在 BCa 中的功能和机制。如 RT-qPCR 所示，circ_0030586 在 BCa 组织和细胞中的表达显着降低。通过琼脂糖凝胶电泳和 RNase R 处理验证了 circ_0030586 的环状特征。集落形成、5-Ethynyl-2'-deoxyuridine 和球体形成测定显示，circ_0030586 的过表达在体外抑制 BCa 细胞增殖和干性。根据异种移植实验，circ_0030586过表达抑制体内肿瘤生长. 从机制上讲，进行了 RNA 下拉和荧光素酶报告基因分析以探索基因之间的相互作用。Circ_0030586 作为 hsa-miR-665 的竞争性内源性 RNA (ceRNA) 上调核受体亚家族 4 A 组成员 3 (NR4A3) 的表达，NR4A3 是 miR-665 在 BCa 中的下游靶基因。MiR-665 在 BCa 组织和细胞中表现出高表达，而 NR4A3 表达在 BCa 中下调。MiR-665 过表达或 NR4A3 沉默逆转了 circ_0030586 过表达对 BCa 细胞增殖和干性的抑制作用。此外，蛋白质印迹分析显示，circ_0030586 通过上调 NR4A3 的表达使细胞外信号调节激酶 (ERK) 通路失活。综上所述，