基因芯片技术筛查并鉴定乳腺癌细胞中MAGE-A11的相关基因

Screening and identification of MAGE-A11 related genes based on DNA microarray

目的:应用高通量基因芯片技术筛查乳腺癌细胞中黑色素瘤相关抗原(melanoma antigen,MAGE)-A11的相关基因,并从数量和功能两方面加以验证。方法:采用基因芯片技术筛选乳腺癌MCF-7、MDA-MB-231和BT-549中MAGE-A11下游靶基因的m RNA的差异表达,对有代表性的基因进行了聚类分析,并利用q RT-PCR进行验证。以CCK-8法、细胞划痕实验和Transwell实验检测MAGE-A11对乳腺癌细胞中增殖、迁移和侵袭功能的影响。结果:3种乳腺癌细胞过表达MAGE-A11导致1 608个下游基因差异表达,主要涉及蛋白泛素化、细胞增殖和凋亡、肿瘤侵袭和转移。基因芯片中典型高表达的ZNF-451、CENPTJ、CDK13、API5和LMO7在q RT-PCR在验证结果中也显著高于对照组(P<0.01),低表达的SHPRH、PML、MARK2、LIMA1和ANGPTL4也显著低于对照组(P<0.01)。转染MAGE-A11组的乳腺癌细胞MCF-7、MDA-MB-231和BT-549 72 h的增殖能力较对照组明显增强(均P<0.01),培养48 h后与对照组相比,转染MAGE-A11的3种细胞划痕出现明显愈合(P<0.05或P<0.01),穿膜数较对照组明显增多(均P<0.01)。结论:在MCF-7、MDA-MB-231和BT-549三种乳腺癌细胞中筛查到涉及蛋白泛素化、细胞增殖和凋亡、肿瘤侵袭和迁移等生物功能众多的表达差异基因,对其中10种典型差异基因从数量和功能两方面进行验证,并得到初步确认。

Objective： To screen related genes of melanoma-associated antigen-A11（MAGE-A11） in breast cancer cells based on highthroughput DNA microarray technology, and to validate from the aspects of quantity and function. Methods： DNA microarray was used to screen the differently-expresseddown-stream m RNAs of MAGE-A11 in breast cancercelllines（MCF-7, MDA-MB-231 and BT-549）.Cluster analysis was applied on representative genes and quantitative RT-PCR was used to validate. CCK-8, scratch wound healing assay and Transwell assaywere used to detect the effect of MAGE-A11 on the proliferation,migration and invasion of breast cancer cells.Results： Over-expression of MAGE-A11 caused the differential expression of 1608 down-stream genes in 3 breast cancer cell lines,which was associated with various cell functions such as protein ubiquitination,cell proliferation and apoptosis, tumor invasion and metastasis.q RT-PCR validated that the expression of ZNF-451, CENPTJ, CDK13, API5 and LMO7, which were highly expressed in microarray, were also significantly higher than those in control group（P〈0.01）;in addition, SHPRH, PML, MARK2, LIMA1 and ANGPTL4,which were low-expressed in microarray, were also significantly lower than those in control group（P〈0.01）. MAGE-A11 transfection directly increased the proliferation of breast cancer MCF-7, MDA-MB-231 and BT-549 cells at 72 h（all P〈0.01）; compared with control group after transfectionexhibited obvious wound healing at 48 h（P〈0.05 or P〈0.01） and significantly increased trans-membrane cell numbers（all P〈0.01）. Conclusion： Many differentially expressed genes related to ubiquitination, cell proliferation and apoptosis, tumorinvasion and migration were screened in MCF-7, MDA-MB-231 and BT-549 breast cancer cells. Among them, 10 typical differentially expressed genes were identified in terms of quantity and function.