Zhong Yanxin, Liu Yu, Tong Weilai, Xie Xinsheng, Nie Jiangbo, Yang Feng, Liu Zhili, Liu Jiaming
Department of Orthopedics, Nanchang 330006, China.
Jiangxi Provincial Key Laboratory of Spine and Spinal Cord Disease, Nanchang 330006, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2024 Dec 20;44(12):2308-2316. doi: 10.12122/j.issn.1673-4254.2024.12.06.
To investigate the regulatory mechanism of aurora kinase B (AURKB) for promoting malignant phenotype of osteosarcoma cells.
HA-Vector or HA-AURKB was transfected in 293T cells to identify the molecules interacting with AURKB using immunoprecipitation combined with liquid chromatography-tandem mass spectrometry followed by verification with co-immunoprecipitation and Western blotting. In cultured osteosarcoma cells with lentivirus-mediated RNA interference of AURKB or DHX9 or their overexpression, the changes in cell proliferation, migration, and invasion activities were observed with EDU and Transwell assays. Mechanistic analysis was performed using Co-IP and ubiquitination experiments to detect the interaction between AURKB and DHX9 and the phosphorylation and ubiquitination levels of DHX9. Western blotting was used to detect the effect of AURKB and DHX9 on activation of nuclear factor-κB (NF-κB) signaling.
AURKB was highly expressed in osteosarcoma cell lines, and in osteosarcoma 143B cells, AURKB silencing significantly reduced cell proliferation, migration and invasion abilities. Interactions between AURKB and DHX9 were detected, and they were both highly expressed in osteosarcoma tissues; silencing AURKB reduced the protein expression of DHX9, and AURKB overexpression increased DHX9 phosphorylation. Silencing AURKB did not significantly affect the transcription and translation of DHX9 but accelerated its degradation and ubiquitination. Overexpression of DHX9 effectively reversed the effects of AURKB silencing on IKBα protein and phosphorylated p65, promoted nuclear translocation of p65 to activate the NF-κB signaling pathway, and enhanced the proliferation, migration, and invasion abilities of cultured osteosarcoma cells.
AURKB overexpression promotes the malignant phenotype of osteosarcoma cells by activating the NF-κB signaling pathway via regulating DHX9.
探讨极光激酶B（AURKB）促进骨肉瘤细胞恶性表型的调控机制。
将HA-载体或HA-AURKB转染至293T细胞中，采用免疫沉淀结合液相色谱-串联质谱法鉴定与AURKB相互作用的分子，随后通过免疫共沉淀和蛋白质免疫印迹法进行验证。在经慢病毒介导的AURKB或DHX9 RNA干扰或过表达的培养骨肉瘤细胞中，采用EdU和Transwell实验观察细胞增殖、迁移和侵袭活性的变化。利用免疫共沉淀和泛素化实验进行机制分析，以检测AURKB与DHX9之间的相互作用以及DHX9的磷酸化和泛素化水平。采用蛋白质免疫印迹法检测AURKB和DHX9对核因子-κB（NF-κB）信号通路激活的影响。
AURKB在骨肉瘤细胞系中高表达，在骨肉瘤143B细胞中，AURKB沉默显著降低细胞增殖、迁移和侵袭能力。检测到AURKB与DHX9之间存在相互作用，二者在骨肉瘤组织中均高表达；沉默AURKB可降低DHX9的蛋白表达，AURKB过表达则增加DHX9的磷酸化。沉默AURKB对DHX9的转录和翻译无显著影响，但加速其降解和泛素化。DHX9过表达有效逆转了AURKB沉默对IκBα蛋白和磷酸化p65的影响，促进p65核转位以激活NF-κB信号通路，并增强培养的骨肉瘤细胞的增殖、迁移和侵袭能力。
AURKB过表达通过调控DHX9激活NF-κB信号通路，促进骨肉瘤细胞的恶性表型。
