Wang Jing, Dai Wang, Zhang Ming
SICU, Children's Hospital of Nanjing Medical University, Nanjing, China.
Int J Neurosci. 2024 May;134(5):530-542. doi: 10.1080/00207454.2022.2126772. Epub 2022 Oct 3.
Neuroblastoma (NB) is a common malignancy occurring in infants and young children. Centrosome-associated protein E (CENPE) is a kinetochore-related motor protein highly expressed in NB, with the mechanism largely unknown. This study is committed to investigating the role and mechanism of CENPE in NB. Short hairpin RNAs targeting CENPE and E2F transcription factor 1 (shCENPE and shE2F1) and CENPE overexpression plasmid were transfected into IMR-32 and SK-N-SH cells. The mRNA expressions of CENPE, N-Cadherin, Vimentin, and proliferating cell nuclear antigen (PCNA) in NB cells were detected by qRT-PCR. The viability, migration, and invasion of cells were tested through cell function experiments. Western blot was applied to detect the protein levels of N-Cadherin, Vimentin, PCNA, CENPE and Forkhead box M1 (FOXM1). The relationship between CENPE and E2F1 was verified by dual-luciferase reporter assay, while the interaction between FOXM1 and CENPE in NB cells was analyzed by rescue experiments. CENPE expression was upregulated in NB cells from metastatic sites. Silencing of CENPE suppressed the NB cell viability, migration, and invasion; and decreased N-Cadherin, Vimentin and PCNA expressions, while overexpressed CENPE did oppositely. E2F1 positively targeted CENPE and CENPE partly reversed the effects of shE2F1 on repressing NB cell viability, migration, invasion and the activation of CENPE/FOXM1 signaling pathway. In addition, silenced FOXM1 partly offset the effects of CENPE on promoting NB cell migration and invasion. E2F1 induces NB cell migration and invasion activating CENPE/FOXM1 pathway.
神经母细胞瘤（NB）是婴幼儿常见的恶性肿瘤。中心体相关蛋白E（CENPE）是一种与动粒相关的运动蛋白，在NB中高表达，其机制 largely unknown。本研究致力于探讨CENPE在NB中的作用及机制。将靶向CENPE和E2F转录因子1的短发夹RNA（shCENPE和shE2F1）以及CENPE过表达质粒转染至IMR-32和SK-N-SH细胞中。通过qRT-PCR检测NB细胞中CENPE、N-钙黏蛋白、波形蛋白和增殖细胞核抗原（PCNA）的mRNA表达。通过细胞功能实验检测细胞的活力、迁移和侵袭能力。采用蛋白质免疫印迹法检测N-钙黏蛋白、波形蛋白、PCNA、CENPE和叉头框M1（FOXM1）的蛋白水平。通过双荧光素酶报告基因检测验证CENPE与E2F1之间的关系，同时通过拯救实验分析NB细胞中FOXM1与CENPE之间的相互作用。转移部位的NB细胞中CENPE表达上调。沉默CENPE可抑制NB细胞的活力、迁移和侵袭；降低N-钙黏蛋白、波形蛋白和PCNA的表达，而过表达CENPE则产生相反的效果。E2F1正向靶向CENPE，CENPE部分逆转了shE2F1对抑制NB细胞活力、迁移、侵袭及CENPE/FOXM1信号通路激活的作用。此外，沉默FOXM1部分抵消了CENPE对促进NB细胞迁移和侵袭的作用。E2F1通过激活CENPE/FOXM1途径诱导NB细胞迁移和侵袭。
