骨肉瘤(OS)是人类中最常见的骨恶性肿瘤。先前的证据表明,circ_0032463是各种癌症中的致癌环状RNA(circularRNA),包括OS。然而,circ_0032463参与OS的分子机制尚不清楚。Circ_0032463,microRNA-145-5p(miR-145-5p),GDNF受体α1(GFRA1),使用实时定量聚合酶链反应(RT-qPCR)确定Wilms肿瘤1相关蛋白(WTAP)水平。细胞增殖,凋亡,迁移,入侵,使用5-乙炔基-2'-脱氧尿苷(EdU)分析血管生成,流式细胞术,transwell,和试管形成测定。进行蛋白质印迹分析以测量基质金属蛋白酶2(MMP2),MMP9、GFRA1和WTAP蛋白水平。miR-145-5p与circ_0032463或GFRA1之间的结合使用双荧光素酶报告基因和下拉测定来确认。还在体内使用异种移植肿瘤模型分析了circ_0032463对OS细胞生长的生物学作用。甲基化RNA免疫沉淀测定验证了WTAP与circ_0032463之间的相互作用。Circ_0032463、GFRA1和WTAP水平升高,miR-145-5p在OS组织和细胞中降低。Circ_0032463缺陷可能会阻碍OS细胞增殖,迁移,入侵,血管生成,促进细胞凋亡。机械上,circ_0032463用作miR-145-5p海绵以增加GFRA1表达。体内证明了circ_0032463敲低对肿瘤细胞生长的抑制作用。此外,N6-甲基腺苷(m6A)修饰促进circ_0032463的生物发生。一起来看,m6A介导的circ_0032463生物发生部分通过调节miR-145-5p/GFRA1轴促进OS细胞恶性生物学行为,提示OS治疗的有希望的分子标记。
Osteosarcoma (OS) is the most frequent bone malignancy in humans. Previous evidence suggest that circ_0032463 is an oncogenic circular RNA (circRNA) in various cancers, including OS. However, the molecular mechanism of circ_0032463 involved in OS is still unclear. Circ_0032463, microRNA-145-5p (miR-145-5p), GDNF receptor alpha 1 (GFRA1), and Wilms tumor 1-associated protein (WTAP) levels were determined using real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation, apoptosis, migration, invasion, and angiogenesis were analyzed using 5-ethynyl-2\'-deoxyuridine (EdU), flow cytometry, transwell, and tube formation assays. Western blot analysis was performed to measure matrix metalloproteinase 2 (MMP2), MMP9, GFRA1, and WTAP protein levels. Binding between miR-145-5p and circ_0032463 or GFRA1 was confirmed using a dual-luciferase reporter and pull-down assay. The biological role of circ_0032463 on OS cell growth was also analyzed using a xenograft tumor model in vivo. Methylated RNA immunoprecipitation assay validated the interaction between WTAP and circ_0032463. Circ_0032463, GFRA1, and WTAP levels were increased, and miR-145-5p was decreased in OS tissues and cells. Circ_0032463 deficiency might hinder OS cell proliferation, migration, invasion, angiogenesis, and promote apoptosis in vitro. Mechanically, circ_0032463 worked as a miR-145-5p sponge to increase GFRA1 expression. Repression of circ_0032463 knockdown on tumor cell growth was proved in vivo. Besides, N6-methyladenosine (m6A) modification facilitates the biogenesis of circ_0032463. Taken together, m6A-mediated biogenesis of circ_0032463 facilitates OS cell malignant biological behavior partly via regulating the miR-145-5p/GFRA1 axis, suggesting a promising molecular marker for OS treatment.