Blocking hsa_circ_0006168 suppresses cell proliferation and motility of human glioblastoma cells by regulating hsa_circ_0006168/miR-628-5p/IGF1R ceRNA axis

hsa_circ_0006168 is an oncogenic circular RNA in esophageal cancer. However, its role remains unclarified in tumor progression of gliomas, especially in glioblastoma (GBM).
Cell counting kit-8 assay, transwell assays, western blotting, and xenograft experiment, as well as colony formation assay and flow cytometry were performed to measure cell proliferation and motility. Expression of hsa_circ_0006168, microRNA (miR)-628-3p, insulin‑like growth factor 1 receptor (IGF1R), and Ras/extracellular signal regulated kinases (Erk)-related proteins were determined by quantitative real-time polymerase chain reaction and western blotting. The physical interaction was confirmed by dual-luciferase reporter assay and RNA pull-down assay.
hsa_circ_0006168 and IGF1R were upregulated, and miR-628-5p was downregulated in human GBM tissues and cells. Functionally, blocking hsa_circ_0006168 and overexpressing miR-628-5p suppressed cell proliferation, migration, invasion, and expression of Vimentin and Snail (mesenchymal markers) in A172 and LN229 cells, accompanied with increased E-cadherin (epithelial marker), decreased colony formation, and promoted apoptosis rate. Silencing miR-628-5p counteracted the suppression of hsa_circ_0006168 deficiency on these behaviors, and restoring IGF1R blocked miR-628-5p-mediated inhibition as well. More importantly, hsa_circ_0006168 knockdown could delay xenograft tumor growth in vivo and lower Ras and phosphorylated Erk1/2 expression in vitro and in vivo. Mechanically, hsa_circ_0006168 targeted and sponged miR-628-5p, and IFG1R was a novel target for miR-628-5p. Inhibiting miR-628-5p could abrogate in vitro role of hsa_circ_0006168 knockdown, and similarly IGF1R upregulation counteracted miR-628-5p role.
Silencing hsa_circ_0006168 might suppress GBM proliferation and motility via serving as competitive endogenous RNA for miR-628-5p and regulating IGF1R/Ras/Erk pathway.

hsa_circ_0006168是食管癌中的致癌环状RNA（circular RNA），但其在胶质瘤尤其是胶质母细胞瘤（glioblastoma, GBM）的肿瘤进展中的作用仍未明确。
本研究采用细胞计数试剂盒-8（Cell counting kit-8, CCK-8）实验、Transwell实验、蛋白质印迹法（western blotting）、异种移植实验，以及集落形成实验与流式细胞术，检测细胞增殖与迁移侵袭能力。通过实时定量聚合酶链式反应（quantitative real-time polymerase chain reaction, qRT-PCR）和蛋白质印迹法，检测hsa_circ_0006168、微小RNA（microRNA, miR）-628-3p、胰岛素样生长因子1受体（insulin-like growth factor 1 receptor, IGF1R）以及Ras/细胞外调节蛋白激酶（extracellular signal regulated kinases, Erk）相关蛋白的表达水平。采用双荧光素酶报告基因实验与RNA下拉实验（RNA pull-down assay）验证二者的物理相互作用。
在人GBM组织与细胞中，hsa_circ_0006168与IGF1R呈高表达状态，而miR-628-5p呈低表达状态。功能实验结果显示，在A172与LN229细胞中，敲低hsa_circ_0006168或过表达miR-628-5p均可抑制细胞增殖、迁移与侵袭，下调间质标志物波形蛋白（Vimentin）与Snail的表达，同时上调上皮标志物E-钙粘蛋白（E-cadherin）的表达，减少细胞集落形成并促进细胞凋亡。敲低miR-628-5p可逆转hsa_circ_0006168缺失对上述细胞行为的抑制作用，而过表达IGF1R则可阻断miR-628-5p介导的抑制效应。更重要的是，体内敲低hsa_circ_0006168可延缓异种移植瘤的生长，且在体内外均可降低Ras与磷酸化Erk1/2的表达水平。机制研究表明，hsa_circ_0006168可靶向结合并海绵吸附miR-628-5p，而IGF1R是miR-628-5p的全新靶基因。抑制miR-628-5p可抵消敲低hsa_circ_0006168的体外生物学效应，而过表达IGF1R同样可逆转miR-628-5p的功能。
敲低hsa_circ_0006168可能通过作为miR-628-5p的竞争性内源RNA（competitive endogenous RNA, ceRNA），调控IGF1R/Ras/Erk信号通路，从而抑制GBM的增殖与侵袭迁移能力。