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

Abstract

Objectives: 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.

Conclusions: AURKB overexpression promotes the malignant phenotype of osteosarcoma cells by activating the NF-κB signaling pathway via regulating DHX9.

Keywords: DHX9; aurora kinase B; osteosarcoma; phosphorylation.

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.