Fan Li, Yang Jianghua, Shen Chong, Wu Zhouliang, Hu Hailong
Tianjin Key Laboratory of Urology, Department of Urology, The Second Hospital of Tianjin Medical University, Tianjin, 300211, China; Department of Urology, The Second People's Hospital of Lianyungang, Lianyungang, 222006, Jiangsu, China.
Tianjin Key Laboratory of Urology, Department of Urology, The Second Hospital of Tianjin Medical University, Tianjin, 300211, China.
Acta Histochem. 2021 Jul;123(5):151745. doi: 10.1016/j.acthis.2021.151745. Epub 2021 Jun 25.
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（circRNAs）在包括膀胱癌（BCa）在内的各种肿瘤发生发展过程中发挥癌基因或肿瘤抑制因子的作用。在本研究中，我们探究了circ_0030586（也称为circABCC4，ATP结合盒亚家族C成员4）在BCa中的功能及机制。逆转录-定量聚合酶链反应（RT-qPCR）结果显示，circ_0030586在BCa组织和细胞中的表达显著降低。通过琼脂糖凝胶电泳和核糖核酸酶R处理验证了circ_0030586的环状特征。集落形成、5-乙炔基-2'-脱氧尿苷和球体形成实验表明，circ_0030586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过表达在体内抑制肿瘤生长。从机制上来说，进行了RNA下拉和荧光素酶报告基因实验以探究基因之间的相互作用。circ_0030586作为hsa-miR-665的竞争性内源RNA（ceRNA），上调核受体亚家族4 A组成员3（NR4A3）的表达，而NR4A3是BCa中miR-665的下游靶基因。miR-665在BCa组织和细胞中高表达，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，蛋白质免疫印迹分析表明，circ_0030586通过上调NR4A3表达使细胞外信号调节激酶（ERK）通路失活。总之，circ_0030586通过作为miR-665的ceRNA上调NR4A3表达从而使ERK信号失活，进而抑制BCa细胞增殖和干性。
