Department of Neurosurgery, The First Affiliated Hospital of Kunming Medical University, Kunming, 650032, Yunnan, China.
Department of Neurosurgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450001, Henan, China.
J Mol Neurosci. 2021 Aug;71(8):1683-1695. doi: 10.1007/s12031-021-01820-y. Epub 2021 Mar 24.
Recent researches reported that several circular RNAs (circRNAs) were associated with the glioblastoma (GBM) progression, while the regulatory role of circPITX1 remains unknown in GBM. The real-time quantitative polymerase chain reaction (RT-qPCR) was used to quantify circPITX1, miR-584-5p, and karyopherin b1 (KPNB1) expression in GBM tissues and cells. The proliferation capability of cells was analyzed by Cell Counting Kit-8 (CCK-8) and colony-forming assays. The matrigel angiogenesis assay was used to assess tube formation in GBM cells. Flow cytometry assays were conducted to evaluate the cell cycle distribution of GBM cells. The migration and invasion assays were assessed by transwell assay. The Western blot assay was employed to quantify the protein expression level in GBM tissues and cells. The targets of circPITX1 and miR-584-5p were confirmed by dual-luciferase reporter and RNA pull-down assays. A xenograft experiment in nude mice was used to assess the functional role of circPITX1 in vivo. CircPITX1 was obviously overexpressed in GBM tissues and cells when compared with negative groups. The functional experiment implied that knockdown of circPITX1 suppressed proliferation, angiogenesis, migration, invasion, and tumor growth in vivo along with induced cell cycle arrest of GBM cells. Furthermore, miR-584-5p was a target gene of circPITX1, and knockdown of miR-584-5p could abolish circPITX1 silencing-induced effects on GBM cells. KPNB1 was a target gene of miR-584-5p, and functional experiments revealed that overexpression of miR-584-5p repressed proliferation, angiogenesis, migration, invasion, and cell cycle process in GBM cells by targeting KPNB1. Mechanistically, circPITX1/miR-584-5p/KPNB1 axis regulated GBM process via mediating proliferation, angiogenesis, migration, invasion, and cell cycle process of GBM cells.
最近的研究报告称，几种环状 RNA（circRNAs）与胶质母细胞瘤（GBM）的进展有关，而环状 RNA PITX1（circPITX1）在 GBM 中的调控作用尚不清楚。实时定量聚合酶链反应（RT-qPCR）用于定量 GBM 组织和细胞中 circPITX1、miR-584-5p 和核孔蛋白 b1（KPNB1）的表达。细胞计数试剂盒-8（CCK-8）和集落形成实验分析细胞的增殖能力。Matrigel 血管生成实验用于评估 GBM 细胞的管状形成。流式细胞术分析用于评估 GBM 细胞的细胞周期分布。Transwell 测定用于评估 GBM 细胞的迁移和侵袭能力。Western blot 测定用于定量 GBM 组织和细胞中的蛋白表达水平。双荧光素酶报告和 RNA 下拉实验证实了 circPITX1 和 miR-584-5p 的靶标。裸鼠异种移植实验用于评估 circPITX1 在体内的功能作用。与阴性对照组相比，GBM 组织和细胞中 circPITX1 明显过表达。功能实验表明，circPITX1 敲低抑制 GBM 细胞的增殖、血管生成、迁移、侵袭和体内肿瘤生长，并诱导 GBM 细胞的细胞周期停滞。此外，miR-584-5p 是 circPITX1 的靶基因，而 miR-584-5p 的敲低可以消除 circPITX1 沉默对 GBM 细胞的影响。KPNB1 是 miR-584-5p 的靶基因，功能实验表明，过表达 miR-584-5p 通过靶向 KPNB1 抑制 GBM 细胞的增殖、血管生成、迁移、侵袭和细胞周期进程。机制上，circPITX1/miR-584-5p/KPNB1 轴通过调节 GBM 细胞的增殖、血管生成、迁移、侵袭和细胞周期过程来调节 GBM 过程。
