MAGE-A9对人乳腺癌MDA-MB-231细胞中P53转录活性及功能的影响

Effect of melanoma-associated antigen-A9 on transcriptional activity and function of P53 in human breast cancer MDA-MB-231 cells

目的:探讨黑素瘤相关抗原-A9(melanoma-associated antigen,MAGE-A9)对人乳腺癌细胞中P53转录活性及功能的影响。方法:通过LipofectamineTM2000体外转染质粒pcDNA3.0、pcDNA3.0-p53、pCMV6-MAGE-A9和pcDNA3.0-p53/MAGE-A9至人乳腺癌MDA-MB-231细胞,RT-PCR和Western blotting检测细胞中p21WAF1mRNA和蛋白的表达,荧光素酶报告基因分析检测细胞中p21WAF1启动子介导的荧光素酶表达活性,MTT法检测转染不同质粒对MDA-MB-231细胞增殖的影响。结果:转染pcDNA3.0-p53/MAGE-A9组MDA-MB-231细胞中p21WAF1mRNA和蛋白表达水平均明显低于pcDNA3.0-p53组[(0.15±0.01)vs(0.18±0.02),(0.03±0.00)vs(0.06±0.01);均P<0.05]。转染pcDNA3.0-p53质粒可以增强MDAMB-231细胞中p21WAF1启动子介导的荧光素酶的表达[(58.56±3.47)vs(1.00±0.12),P<0.01],转染pcDNA3.0-p53/MAGE-A9后,MDA-MB-231细胞中p21WAF1启动子介导的荧光素酶的表达较转染pcDNA3.0-p53组明显降低[(22.02±4.91)vs(58.56±3.47),P<0.05]。与pcDNA3.0组相比,pcDNA3.0-p53组MDA-MB-231细胞增殖率明显明显降低[(228.89±22.39)%vs(337.23±23.67)%,P<0.05];而pcDNA3.0-p53/MAGE-A9组MDA-MB-231细胞增殖率明显高于pcDNA3.0-p53组[(291.51±5.91)%vs(228.89±22.39)%,P<0.05]。结论:MAGE-A9可抑制MDA-MB-231细胞中P53的转录活性及细胞增殖。

Objective ： To explore the effect of melanoma-associated antigen （MAGE）-A9 on the transcriptional activity and the function of P53. Methods： Plasmids pcDNA3.0, pcDNA3.0-p53 and pCMV6-MAGE-A9 were transfeced in vitro into human breast cancer MDA-MB-231 cells using LipofectamineTM2000. The expressions of p21WAF1 mRNA and protein in MDA-MB-231 cells were analyzed by RT-PCR and Western blotting. Luciferase reporter assay was performed to determine the luciferase activity induced by p21WAF1 promoter in MDA-MB-231 cells. MTT assay were adopted to explore the effect of different plasmids on the cell proliferation. Results： The expression of p21WAF1 both in mRNA and protein levels was decreased in pcDNA3.0-p53/MAGE-A9 group, compared with pcDNA3.0-p53 group （／[0.15±0.01／] vs ／[0.18±002／], ／[0.03±0.00／] vs ／[0.06±0.01／], P〈0.05）. The luciferase activity induced by p21WAF1 promoter was remarkably increased in MDA-MB-231 cells transfected with plasmid pcDNA3.0-p53 （／[58.56±3.47／] vs ／[1.00±012／], P〈001）. After transfected with pcDNA3.0-p53/MAGE-A9, the luciferase activity induced by p21WAF1 promoter in MDA-MB-231 cells was significantly reduced compared with that in pcDNA3.0-p53 group （／[22.02±4.91／] vs ／[58.56±3.47／], P〈005）. Compared with pcDNA3.0 group, the proliferation rate were significantly decreased in pcDNA3.0-p53 group （／[228.89±22.39／]% vs ／[337.23±23.67／]%, P〈0.05）, while in pcDNA3.0-p53/MAGE-A9 group, it was significantly higher than that in pcDNA3.0-p53 group （／[291.51±5.91／]% vs ／[228.89±22.39／]%, P〈0.05）. Conclusion： MAGE-A9 can inhibit the transcriptional activity of P53 and proliferation of MDA-MB-231 cells.