论文部分内容阅读
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 mitogen-activated checkpoint protein BubR1 in esophageal cancer. Methods The promoter of Bub1b gene was subcloned into vector pSEAP2-Basic to construct recombinant plasmid pSEAP2-Bub1b-P2000. The recombinant plasmid pSEAP2-Bub1b-P2000 was transfected into HEK-293 cells overexpressing c-myc via Ad-c -myc infection) and ECA-109 cells. After 6 h of transfection, ECA-109 cells were treated with c-myc inhibitor 10058-F4. SEP chemiluminescence method was used to detect the ALP activity in each group. The mRNA and protein levels of Bub1b mRNA and protein were detected by RT-PCR and Western blot respectively in ECA-109 cells with suppressed c-myc expression. Results The recombinant plasmids were constructed correctly by PCR, double enzyme digestion and sequencing. The recombinant plasmids transfected HEK-293 cells overexpressing c-myc and ALP activity of EC-109 cells inhibiting c-myc expression were significantly increased (P <0.0001 ) Inhibited the expression of Bub1b mRNA and BubR1 protein in ECA-109 cells by inhibiting the expression of c-myc (P <0.05). Conclusion The oncogene c-myc regulates the expression of BubR1 in esophageal cancer cells, which lays the foundation for further study on the role of BubR1 in esophageal cancer.