AURKB通过DHX9激活NF-κB信号通路促进骨肉瘤细胞恶性表型

doi:10.12122/j.issn.1673-4254.2024.12.06

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (12): 2308-2316.doi: 10.12122/j.issn.1673-4254.2024.12.06

• • 上一篇

AURKB通过DHX9激活NF-κB信号通路促进骨肉瘤细胞恶性表型

钟彦鑫¹^,²(), 刘宇¹^,²(), 童未来¹^,², 谢新生³, 聂江波¹^,², 杨枫¹^,², 刘志礼¹^,², 刘家明¹^,²()

^1.南昌大学第一附属医院，骨科，江西南昌 330006
^3.南昌大学第一附属医院，医学研究公共服务中心，江西南昌 330006
^2.脊柱脊髓系统疾病江西省重点实验室，江西南昌 330006

收稿日期:2024-07-17 出版日期:2024-12-20 发布日期:2024-12-26
通讯作者: 刘家明 E-mail:zyx1998033@163.com;ly18296389906@yeah.net;liujiamingdr@hotmail.com
作者简介:钟彦鑫，在读硕士研究生，E-mail: zyx1998033@163.com
刘宇，在读硕士研究生，E-mail: ly18296389906@yeah.net
第一联系人：钟彦鑫、刘宇共同第一作者
基金资助:
江西省“双千计划”首批培养类项目;江西省自然科学基金(20192ACBL21041)

High expression of AURKB promotes malignant phenotype of osteosarcoma cells by activating nuclear factor-κB signaling via DHX9

Yanxin ZHONG¹^,²(), Yu LIU¹^,²(), Weilai TONG¹^,², Xinsheng XIE³, Jiangbo NIE¹^,², Feng YANG¹^,², Zhili LIU¹^,², Jiaming LIU¹^,²()

^1.Department of Orthopedics, Nanchang 330006, China
^3.Medical Research Public Service Center, First Affiliated Hospital of Nanchang University, Nanchang 330006, China, Nanchang 330006, China
^2.Jiangxi Provincial Key Laboratory of Spine and Spinal Cord Disease, Nanchang 330006, China

Received:2024-07-17 Online:2024-12-20 Published:2024-12-26
Contact: Jiaming LIU E-mail:zyx1998033@163.com;ly18296389906@yeah.net;liujiamingdr@hotmail.com

摘要/Abstract

摘要：

目的探讨AURKB在促进骨肉瘤细胞恶性表型中的具体调控机制。方法分别转染HA-Vector和HA-AURKB于293T细胞中，采用免疫沉淀结合液相色谱-串联质谱技术筛选AURKB的潜在互作分子，免疫共沉淀和Western blotting验证其互作分子。通过RNA干扰技术，干扰骨肉瘤细胞中AURKB的表达；设置对照组：转染对照组慢病毒143B细胞，sh-AURKB组：转染干扰AURKB慢病毒143B细胞，sh-con+Flag-DHX9组：对照组转染过表达DHX9慢病毒，sh-AURKB+Flag-DHX9组：沉默AURKB组转染过表达DHX9慢病毒。采用EDU、Transwell实验分别观测细胞的增殖、迁移和侵袭活性。机制分析采用Co-IP、体内泛素化实验等检测AURKB和DHX9的相互作用、DHX9磷酸化水平、DHX9泛素化水平。Western blotting检测AURKB及DHX9对NF-κB的信号激活作用。结果 AURKB在骨肉瘤细胞系高表达。沉默骨肉瘤143B细胞中AURKB的表达，细胞增殖、迁移和侵袭能力下降（P<0.05）。而AURKB与DHX9存在相互作用，且AURKB及DHX9在骨肉瘤组织中均高表达，沉默AURKB能够降低DHX9的蛋白表达，过表达AURKB后DHX9磷酸化水平升高（P<0.05）。沉默AURKB后不影响DHX9的转录翻译，但可以加快DHX9降解速度及增加DHX9泛素化水平。此外，过表达DHX9可以逆转沉默AURKB对IKBα蛋白及磷酸化p65的影响，促进p65入核激活NF-κB信号通路，并且增强了骨肉瘤细胞增殖、迁移和侵袭能力。结论 AURKB通过调控DHX9激活NF-κB信号通路，促进骨肉瘤细胞恶性表型。

关键词: 骨肉瘤, AURKB, DHX9, 磷酸化

Abstract:

Objective To investigate the regulatory mechanism of aurora kinase B (AURKB) for promoting malignant phenotype of osteosarcoma cells. Methods 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 in vivo 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. Results 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. Conclusion AURKB overexpression promotes the malignant phenotype of osteosarcoma cells by activating the NF-κB signaling pathway via regulating DHX9.

Key words: osteosarcoma, aurora kinase B, DHX9, phosphorylation

钟彦鑫, 刘宇, 童未来, 谢新生, 聂江波, 杨枫, 刘志礼, 刘家明. AURKB通过DHX9激活NF-κB信号通路促进骨肉瘤细胞恶性表型[J]. 南方医科大学学报, 2024, 44(12): 2308-2316.

Yanxin ZHONG, Yu LIU, Weilai TONG, Xinsheng XIE, Jiangbo NIE, Feng YANG, Zhili LIU, Jiaming LIU. High expression of AURKB promotes malignant phenotype of osteosarcoma cells by activating nuclear factor-κB signaling via DHX9[J]. Journal of Southern Medical University, 2024, 44(12): 2308-2316.

图/表 4

图1 沉默AURKB抑制骨肉瘤143B细胞的增殖、迁移和侵袭能力

Fig.1 Silencing AURKB inhibits proliferation, migration and invasion of osteosarcoma 143B cells. A: Survival curves of sarcoma patients with different AURKB expression levels. B: Western blotting for detecting AURKB expression levels in osteosarcoma cell lines and for verifying the efficiency of AURKB knockdown in 143B cells. C: EDU assay for detecting the effect of AURKB silencing on proliferation of osteosarcoma cells (Original magnification: ×100). D: Migration and invasion assays of osteosarcoma cells after silencing AURKB (×100). *P<0.05,**P<0.01 vs sh-con.

图2 AURKB与DHX9存在相互作用

Fig2 AURKB interacts with DHX9. A: DHX9 peptide identified by mass spectrometry. B: Silver-staining assay reveals AURKB-bound proteins. C: CO-IP detection of AURKB and DHX9 interaction in HEK-293 cells. D: CO-IP detection of AURKB and DHX9 interaction in 143B cells. E: Protein expression levels of AURKB and DHX9 in 8 pairs of osteosarcoma tissues and adjacent tissues (T: Tumor; N: Normal). **P<0.01.

图3 AURKB磷酸化DHX9抑制其泛素化降解

Fig.3 AURKB phosphorylates DHX9 to inhibit its ubiquitinated degradation. A: Survival curves of osteosarcoma patients with low and high AURKB expression. B: Expression of DHX9 in osteosarcoma cell lines. C: Impact of AURKB knockdown on DHX9 protein expression. D: Co-IP analysis of DHX9 protein phosphorylation levels following AURKB overexpression (The diagonal lines represent paired analyses from the same set of experiments). E: DHX9 mRNA expression levels in 143B cells with AURKB silencing. F: Time-dependent analysis of DHX9 protein expression in AURKB-silenced 143B cells treated with cycloheximide. G: Levels of DHX9 and AURKB in AURKB-knockdown 143B cell treated with MG132 (20 μmol/L) or chloroquine (CQ, 20 μmol/L) for 12 h detected by Western blotting. H: Ubiquitination levels of DHX9 in AURKB-knockdown 143B cells. *P<0.05, **P<0.01 vs sh-con.

图4 AURKB通过调控DHX9激活NF-κB信号通路促进骨肉瘤细胞恶性表型

Fig.4 High expression of AURKB enhances malignant phenotype of osteosarcoma cells by activating NF-κB signaling via regulating DHX9. A: EDU experiment for assessing the effects of AURKB silencing and DHX9 overexpression on proliferation of osteosarcoma cells (×100). B: Migration and invasion experiments for assessing the effects of AURKB silencing and DHX9 overexpression on migration and invasion of osteosarcoma cells (×100). C: Western blotting for detecting protein levels of IKBα, p65, phosphorylated IKBα, and phosphorylated p65 in osteosarcoma cells with AURKB silencing and DHX9 overexpression. D: Western blotting for assessing the impact of AURKB silencing and DHX9 overexpression on nuclear translocation of p65. *P<0.05, **P<0.01, ***P<0.001 vs sh-con+Flag-Vector. #P<0.05, ##P<0.01 vs sh-AURKB+Flag-Vector.

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AURKB通过DHX9激活NF-κB信号通路促进骨肉瘤细胞恶性表型

High expression of AURKB promotes malignant phenotype of osteosarcoma cells by activating nuclear factor-κB signaling via DHX9

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