[Aurora kinase-B silencing promotes apoptosis of osteosarcoma 143B cells by ULK1 phosphorylation-induced autophagy].

ABSTRACT: OBJECTIVE:To investigate the effect of Aurora kinase B (AURKB) silencing-induced autophagy on apoptosis of osteosarcoma 143B cells and the underlying molecular mechanisms. METHODS:Human osteosarcoma 143B cells were transfected with Lv/shAURKB or the negative control vector Lv/shScrambled followed by treatment with chloroquine (CQ) for 24 h. Western blotting was performed to detect the protein expression levels of AURKB, P62, LC3, cleaved caspase-3, Bcl-2, and P-ULK1Ser555. Transmission electron microscopy and LC3 dual-label fluorescence method were used to trace the autophagosomes in 143B cells to assess cell autophagy, and the cell apoptosis was detected using flow cytometry and TUNEL assay. Co-immunoprecipitation assay was used to detect the interaction between AURKB and ULK1. RESULTS:The ratio of autophagy-related proteins LC3 II/I and the number of autophagosomes were significantly increased in 143B cells after transfection with Lv/shAURKB (P < 0.05), which significantly increased the expression of cleaved caspase-3 and reduced the expression of Bcl-2 (P < 0.05). Combined treatment of the cells with Lv/shAURKB and the autophagy inhibitor chloroquine obviously restored the expressions of caspase-3 and Bcl-2 (P < 0.05). Transfection with Lv/shAURKB significantly increased the apoptosis rate of 143B cells (P < 0.05), and this effect was significantly antagonized by combined treatment with chloroquine (P < 0.05). AURKB silencing strongly activated the phosphorylation of the autophagy-initiating protein ULK1Ser555 in 143B cells (P < 0.05). The results of co-immunoprecipitation assay confirmed when AURKB was immunoprecipitated, ULK1 also precipitated. CONCLUSIONS:Silencing AURKB can induce autophagy by activating ULK1Ser555 phosphorylation to promote apoptosis in 143B cells.

SUBMITTER: Wu X 

PROVIDER: S-EPMC7544570 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature