C-myc通过调控BubR1影响食管鳞癌细胞对紫杉醇的敏感性

中国生物工程杂志

2013, Vol. 33

Issue (4): 22-27

研究报告

C-myc通过调控BubR1影响食管鳞癌细胞对紫杉醇的敏感性

胡敏, 宋培培, 湛晓琴, 吕自兰, 陈楚, 施琼, 翁亚光

重庆医科大学医学检验系临床检验诊断学教育部重点实验室重庆 400016

C-Myc Affects Esophageal Squamous Cancer Sensitivity to Paclitaxel by Regulating BubR1 Expression

HU Min, SONG Pei-pei, ZHAN Xiao-qin, LÜ Zi-lan, CHEN Chu, SHI Qiong, WENG Ya-guang

The Key Laboratory of Laboratory Medical Diagnostics in Ministry of Education, Chongqing Medical University, Chongqing 400016, China

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摘要： 探讨C-Myc与有丝分裂期检查点蛋白BubR1的表达关系和对紫杉醇药物作用的可能影响。用免疫组化方法检测23例食管鳞癌组织标本中C-Myc和BubR1的表达水平,并通过免疫印迹的方法比较3株食管鳞癌细胞株ECA-109,KYSE150和KYSE180中C-Myc和BubR1的表达高低,分析相关性;将人BUB1b基因启动子上游约2000bp片段插入pSEAP2分泌型碱性磷酸酶报告质粒中构建为pSEAP2-BubR1-P2000,分别转染至3株鳞癌细胞内,检测启动子激活效果;在ECA-109细胞内过表达C-Myc后再转染pSEAP2-BubR1-P2000后,检测启动子的激活效果;免疫印迹方法检测C-Myc抑制剂10058-F4对BubR1蛋白表达的影响;通过MTT检测10058-F4干扰C-Myc后ECA-109细胞在梯度紫杉醇浓度下的生存率变化;最后通过DAPI染色观察单用C-Myc抑制剂,单用低浓度紫杉醇(100nM)或联用10058-F4和紫杉醇组的凋亡比例。结果发现在临床食管鳞癌标本和食管鳞癌细胞株中C-Myc和BubR1的表达有相关一致性;C-Myc高表达的细胞株中BubR1启动子活性的激活程度更强,并且过表达C-Myc后能进一步上调启动子活性;C-Myc特异性抑制剂10058-F4可以有效下调BubR1表达,并减低细胞在梯度紫杉醇作用下的细胞生存率。DAPI染色结果显示联用10058-F4和低浓度紫杉醇能明显增加细胞处于有丝分裂期的比率。在食管鳞癌细胞中C-Myc能上调有丝分裂期检查点蛋白BuR1的水平,并与食管癌对紫杉醇的敏感性相关,C-Myc可能通过上调BubR1表达而减低食管鳞癌对紫杉醇的反应。

关键词： C-Myc; 有丝分裂期检查点蛋白BubR1; C-Myc抑制剂10058-F4; 紫杉醇

Abstract: To explore the relationship between C-Myc and mitotic checkpoint protein BubR1 expression and possible influence of C-Myc on paclitaxel drug effects. Immunohistochemistry assay was explored to detect C-Myc and BubR1 expression in twenty-three clinical esophageal squamous cancer samples. And Western blot was applied to detect and compare C-Myc and BubR1 expression levels in three esophageal squamous cancer cell lines, ECA-109, KYSE150 and KYSE180. The correlation between C-Myc and BubR1 protein expression was analyzed. About 2000bp fragment upstream human BUB1b gene was cloned and inserted into pSEAP2 promoter reporter vector to construct pSEAP2-BubR1-P2000. Then pSEAP2-BubR1-P2000 was transfected into three cell lines for promoter activities detection (ALP activity). ALP activity was detected in ECA-109 cells both overexpressing C-Myc and transfected pSEAP2-BubR1-P2000. Western blot assay was used to detect the effect of C-Myc inhibitor 10058-F4 on BubR1 expression. MTT assay was applied to detect cell viability under gradient paclitaxel exposure after C-Myc suppression. DAPI staining was explored for apoptotic cell count in C-Myc inhibitor group, low concentration paclitaxel (100nM) group and combination of C-Myc inhibitor and paclitaxel, respectively. The results showed that C-Myc expression positively correlated with BubR1 expression both in clinical esophageal squamous cancer tissues and in esophageal squamous cancer cell lines. Cells with high C-Myc expression showed higher BubR1 promoter activity and overexpression of C-Myc in ECA-109 cells can further enhance BubR1 promoter activity. Specific C-Myc inhibitor 10058-F4 can effectively down-regulate BubR1 expression and decrease cell viability under gradient paclitaxel exposure. DAPI staining result exhibited that combination of 10058-F4 and paclitaxel induced significantly more mitotic cells than single usage of 10058-F4 or paclitaxel. In esophageal squamous cancer cells, C-Myc can regulate mitotic checkpoint protein BubR1 expression and related with paclitaxel sensitivity. C-Myc may reduce esophageal squamous cancer response to paclitaxel by up-regulating BubR1 expression.

Key words: C-Myc Mitotic checkpoint protein BubR1 C-Myc inhibitor 10058-F4 Pacliaxel

收稿日期: 2012-10-15 出版日期: 2013-04-25

ZTFLH:

Q819

基金资助: 教育部高等学校博士学科点专项科研基金资助项目(20115503110009)

通讯作者: 翁亚光 E-mail: yaguangweng@126.com

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引用本文:

胡敏, 宋培培, 湛晓琴, 吕自兰, 陈楚, 施琼, 翁亚光. C-myc通过调控BubR1影响食管鳞癌细胞对紫杉醇的敏感性[J]. 中国生物工程杂志, 2013, 33(4): 22-27.

HU Min, SONG Pei-pei, ZHAN Xiao-qin, LÜ Zi-lan, CHEN Chu, SHI Qiong, WENG Ya-guang. C-Myc Affects Esophageal Squamous Cancer Sensitivity to Paclitaxel by Regulating BubR1 Expression. China Biotechnology, 2013, 33(4): 22-27.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I4/22

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