抑制Aurora激酶B的表达可促进骨肉瘤143B细胞凋亡

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

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ULK1^Ser555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ULK1^Ser555磷酸化诱导细胞自噬促进骨肉瘤143B细胞凋亡。

Objective To investigate the effect of Aurora kinase B(AURKB) silencing-induced autophagy on apoptosis of osteosarcoma 143 B cells and the underlying molecular mechanisms.Methods Human osteosarcoma 143 B cells were transfected with Lv/shAURKB or the negative control vector Lv/shScrambled followed by treatment with chloroquine(CQ) for24 h.Western blotting was performed to detect the protein expression levels of AURKB,P62,LC3,cleaved caspase-3,Bcl-2,and P-ULK1^Ser555.Transmission electron microscopy and LC3 dual-label fluorescence method were used to trace the autophagosomes in 143 B 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 143 B 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 143 B 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 143 B 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 ULK1^Ser555 phosphorylation to promote apoptosis in 143 B cells.