癌基因c-myc对食管癌BubR1的调控  

Regulatory effect of oncogene c-myc on mitotic checkpoint protein BubR1 in esophageal cancer

作　　者：宋培培[1] 胡敏[1] 湛晓琴[1] 章帆[1] 施琼[1] 翁亚光[1] 

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016

出　　处：《中国生物制品学杂志》2013年第12期1758-1763,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金资助项目(30872770);教育部高等学校博士学科点专项科研基金(20115503110009)

摘　　要：目的探讨癌基因c-myc对食管癌有丝分裂检查点效应蛋白BubR1的调控作用。方法将Bub1b基因启动子亚克隆至载体pSEAP2-Basic中,构建重组质粒pSEAP2-Bub1b-P2000,在Lipofectamine 2000的介导下,分别转染过表达c-myc的HEK-293细胞(经Ad-c-myc感染)和ECA-109细胞;转染6 h后,经c-myc抑制剂10058-F4作用ECA-109细胞。SEAP化学发光法检测各组细胞的ALP活性;采用RT-PCR和Western blot法分别检测抑制c-myc表达的ECA-109细胞中Bub1b基因mRNA转录和BubR1蛋白的表达水平。结果重组质粒经PCR、双酶切及测序鉴定构建正确;重组质粒转染过表达c-myc的HEK-293细胞和抑制c-myc表达ECA-109细胞的ALP活性明显升高(P<0.000 1);抑制c-myc表达可明显下调ECA-109细胞中Bub1b基因mRNA转录和BubR1蛋白的表达水平(P<0.05)。结论癌基因c-myc对食管癌细胞中BubR1的表达有正调控作用,为进一步研究BubR1在食管癌中发挥的作用奠定了基础。Objective To investigate the regulatory effect of oncogene c-myc on the mitotic checkpoint protein BubRl in esophageal cancer. Methods The Bublb gene promoter was subcloned into vector pSEAP-Basic, and the constructed recombinant plasmid pSEAP2-Bublb-P2000 was transfected to HEK-293 cells transfected with Ad-c-myc, in which c-myc was over- expressed, in mediation of Lipofeetamine 2000. ECA-109 ceils were transfeeted with the recombinant plasmid and treated with c-myc inhibitor 10058-F4 6 h after transfection. The cells in various groups were determined for ALP activity by SEAP chemiluminescence immunoassay. The Bublb mRNA transcription and BubR1 protein expression levels in ECA-109 cells were determined by PCR and Western blot respectively. Results PCR, restriction analysis and sequencing proved that recombinant plasmid pSEAP2-Bublb-P2000 was constructed correctly. Both the ALP activities of HEK-293 cells and ECA- 109 cells increased significantly （P 〈 0. 000 1 ）, while inhibition of c-myc down-regulated the Bub lb mRNA transcription and BubRl protein expression levels of BubR1 significantly （P 〈 0. 05）. Conclusion Oncogene c-myc positively regulated the expression of B ubR 1 in esophageal cancer cells, which laid a foundation of further study on role of B ubR 1 in esophageal cancer.

关 键 词：C-MYC基因 有丝分裂检查点 BubR1蛋白 食管癌 

分 类 号：R735.1[医药卫生—肿瘤]