南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (09): 1273-1279.doi: 10.12122/j.issn.1673-4254.2020.09.08

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抑制Aurora激酶B的表达可促进骨肉瘤143B细胞凋亡

吴 昕，刘家明，宋宏海，杨起坤，应 辉，刘志礼   

  • 出版日期:2020-09-20 发布日期:2020-09-20

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

  • Online:2020-09-20 Published:2020-09-20

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摘要： 目的 探讨抑制Aurora激酶B（AURKB）诱导骨肉瘤143B细胞自噬对凋亡的影响及其潜在的分子机制。方法 构建慢病毒载体（干扰慢病毒Lv/shAURKB和相应的空载慢病毒Lv/shScrambled），分别感染人源骨肉瘤细胞系143B细胞，并采用氯喹（CQ）进行干预24 h，分别作为：AURKB慢病毒干扰组（plv/shAURKB组）；阴性对照组（plv/NC组）；plv/shAURKB+CQ组；空载慢病毒+氯喹处理组（plv/NC+CQ组）。RT-qPCR检测Lv/shAURKB慢病毒载体对AURKB mRNA的干扰效率。Western blot检测AURKB、P62、LC3、Cleaved-Caspase3、Bcl2、P-ULK1（Ser555）等蛋白表达水平。采用透射电镜和LC3双标荧光法示踪143B细胞内自噬小体并检测自噬水平，流式细胞术和Tunel实验检测细胞凋亡。免疫共沉淀检测AURKB蛋白与ULK1（UNC-51样激酶1）蛋白间的相互作用关系。结果 plv/shAURKB组细胞中AURKB mRNA及蛋白表达水平均低于plv/NC组，差异有统计学意义（P<0.05）；plv/shAURKB组中自噬相关蛋白LC3Ⅱ/Ⅰ的比率和自噬小体数量高于plv/NC组，而P62的表达低于plv/NC组，差异均有统计学意义（P<0.05）；plv/shAURKB组细胞中促凋亡蛋白Cleaved-caspase3显著高于plv/NC组（P<0.05），而抑制凋亡蛋白Bcl2的表达水平显著低于plv/NC组（P<0.05）；与plv/shAURKB组相比plv/shAURKB+CQ组中凋亡相关蛋白 Cleaved-caspase3和Bcl2的表达得到明显回复（P<0.05）。Tunel实验和流式细胞术结果显示，plv/shAURKB组细胞凋亡率显著高于plv/NC组（P<0.05），plv/shAURKB+CQ组细胞凋亡率与plv /shAURKB组相比得到明显的回复（P<0.05）。plv/shAURKB组细胞中自噬启动蛋白ULK1Ser555磷酸化水平显著高于plv/NC组（P<0.05）。免疫共沉淀检测显示：免疫沉淀AURKB的同时ULK1也发生了沉淀。结论 沉默AURKB能够通过激活ULK1Ser555磷酸化诱导细胞自噬促进骨肉瘤143B细胞凋亡。

关键词: 骨肉瘤, Aurora激酶B, ULK1, 自噬, 凋亡

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. Conclusion Silencing AURKB can induce autophagy by activating ULK1Ser555 phosphorylation to promote apoptosis in 143B cells.

Key words: osteosarcoma, Aurora kinase B, ULK1 protein, autophagy, apoptosis