阿立塞替通过靶向Aurora激酶A提高人非小细胞肺癌细胞的放射敏感性

Study on Alisertib Enhancing Radiosensitivity of Human Non-small Cell Lung Cancer Cells by Targeting Aurora Kinase A

目的:探讨阿立塞替(MLN8237)对人非小细胞肺癌放射敏感性的影响及作用机制。方法:用siPORT NeoFX转染试剂将阴性siRNA、si Aurora Kinase A分别转染NCI-H1975细胞阴性对照组(Si control)及靶向Aurora激酶A组(si Aurora Kinase A),未转染siPORT NeoFX的NCI-H1975细胞作为单纯对照组(Control);CCK8实验检测抑制Aurora激酶A的抑制剂(MLN82370)对NCI-H1975细胞生长的影响,细胞凋亡实验检测抑制Aurora激酶A及加入MLN8237对NCI-H1975细胞凋亡的影响,克隆形成实验检测了MLN8237对NCI-H1975细胞放射敏感性的影响影响,qRT-PCR实验评估了NCI-H1975细胞中p16及p21的表达,荧光素酶分析实验测定细胞周期相关转录因子(E2F)和应激活化蛋白激酶(JNK)的活性。结果:NCI-H1975细胞的转染效率达89.91%,抑制Aurora激酶A及加入MLN8237可明显抑制NCI-H1975细胞的生长,差异有统计学意义(P<0.01);细胞凋亡实验结果显示,抑制Aurora激酶A及加入MLN8237可明显提高NCI-H1975细胞的凋亡率;克隆形成实验显示,MLN8237能够显著降低照射后NCI-H1975细胞的克隆形成能力对NCI-H1975细胞有放射增敏效果,且随剂量增大其增敏效果越显著;qRT-PCR结果显示,加入MLN8237(Aurora激酶A抑制剂)照射后,p16及p21的表达明显上调;荧光素酶分析实验结果显示,抑制Aurora激酶A显著降低了E2F的活性,而JNK的活性显著提高,差异均有统计学意义(P<0.01)。结论:阿立塞替能通过靶向Aurora激酶A,而抑制NCI-H1975细胞增殖及诱导其凋亡,从而提高NCI-H1975细胞的放射敏感性。

Objective:To investigate the effect of alisertib on the radiosensitivity of human non-small cell lung cancer by targeting Aurora kinase A.Methods:NCI-H1975 cells were transfected with siPORT NeoFX transfection reagent and divided into Si control group,Si Aurora Kinase A group;Nci-H1975 cells that were not transfected with siPORT NeoFX were used as the control group.The effects of inhibitors that inhibit Aurora kinase A(MLN82370)on NCI-H1975 cells growth were detected by CCK8 assay;the effects of Aurora kinase A inhibition and the addition of MLN8237 on the apoptosis of NCI-H1975 cells were detected by apoptosis assay;the effects of MLN8237 on radiosensitivity of NCI-H1975 cells were examined by cloning formation experiment;the expression of p16 and p21 in NCI-H1975 cells was evaluated by qRT-PCR;the activities of E2F and JNK were determined by luciferase assay.Results:The transfection efficiency of NCI-H1975 cells was 89.91%;CCK8 results showed the growth of NCI-H1975 cells were significantly inhibited by Aurora kinase A or adding the MLN8237,the difference was statistically significant(P<0.01).The results of apoptosis assay showed the apoptosis of NCI-H1975 cells were significantly increased by inhibiting of Aurora kinase A or adding MLN8237;The cloning formation experiments showed the cloning forming ability of NCI-H1975 cells were significantly reduced with MLN8237 after irradiation,the radiosensitivity of NCI-H1975 cells were significantly increased with MLN8237;The qRT-PCR results showed the expression of p16 and p21 was significantly up-regulated after IR with MLN8237(Aurora kinase A inhibitor);The results of Luciferase analysis showed that the activity of E2F was significantly decreased while the activity of JNK was significantly increased by inhibition of Aurora kinase A(P<0.01).Conclusion:Alisertib can inhibit the proliferation and induce apoptosis of NCI-H1975 cells by targeting Aurora kinase A,thereby increasing the radiosensitivity of NCI-H1975 cells.