人黑素瘤抗原MAGE-A4对p53转录活性的影响

Effect of melanoma antigen MAGE-A4 on the transcriptional activity of p53

目的:探讨人黑素瘤抗原-A(melanoma antigen-A,MAGE-A)对p53转录活性的影响。方法:选用p53缺失型人肺癌细胞系H1299,采用荧光素酶报告分析法、RT-PCR、Western印迹法、克隆形成实验和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling,TUNEL)等方法研究MAGE-A4对p53转录活性的影响。结果:荧光素酶报告分析结果显示,转染p53基因(25ng)可以使p21WAF1启动子介导的荧光素酶表达增加(P<0.01);共转染恒定量(25ng)的p53基因和不同量的MAGE-A4基因(100、200和400ng)后,p21WAF1启动子介导的荧光素酶表达均明显高于单独转染p53基因时的表达量(P<0.01)。RT-PCR和Western印迹法检测结果也显示,转染p53基因可以使p21WAF1mRNA和蛋白表达水平增加(P<0.01),共转染MAGE-A4基因和p53基因后,p21WAF1mRNA和蛋白表达水平均明显高于单独转染p53基因时的表达水平(P<0.01)。克隆形成实验及TUNEL染色结果显示,转染p53基因可以使H1299细胞克隆形成数减少及凋亡细胞数增加(P<0.01),共转染MAGE-A4基因和p53基因以后,H1299细胞克隆形成数与单独转染p53基因组相比减少,而凋亡细胞数与单独转染p53基因组比较增加(P<0.01)。结论:MAGE-A4可以增强p53的转录活性及功能发挥。

Objective：To investigate the effect of melanoma antigen A （MAGE-A4） on the transcriptional activity of p53. Methods： Luciferase reporter assay, RT-PCR, Western blotting, colony formation assay and TUNEL （terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling） staining were performed to study the effect of MAGE-A4 on p53 transcriptional activity in p53-deficient human lung carcinoma H1299 cells. Results： Luciferase reporter assay showed that p53 （25 ng） increased the luciferase activities induced by p21WAF1 promotors （P〈0.01）. After co-transfection with the constant amounts of p53 （25 ng） and the increasing amounts of MAGE-A4 （100, 200, and 400 ng）, the luciferase activities induced by p21WAF1 promotors were significantly increased as compared with the group that transfected with p53 （25 ng） only （P〈0.01）. RT-PCR and Western blotting also showed that transfection of p53 increased the mRNA and protein expressions of p21WAF1 （P〈0.01） and the expressions of p21WAF1 were increased after co-transfection with p53 and MAGE-A4 than those transfected with p53 only （P〈0.01）. Colony formation assay and TUNEL staining discovered that the colony numbers were decreased and the apoptotic cell numbers were increased in H1299 cells after transfection with p53 as compared with the control （P〈0.01） and the colony numbers were further decreased and the apoptotic cell numbers were further increased in H1299 cells after co-transfection of p53 and MAGE-A4 as compared with the group that tranfected with p53 alone （P〈0.01）. Conclusion： MAGE A4 enhanced p53 transcriptional activity and functions.