可条件性诱导PLK1稳定敲降的食管癌细胞株的建立

doi:10.3969/j.issn.1004-616x.2014.05.006

癌变·畸变·突变

可条件性诱导PLK1稳定敲降的食管癌细胞株的建立

曹迎亚^1，2，车轶群¹，陈群¹，马文韬¹，刘洋¹，郝佳洁¹，蔡岩¹，鲁卫华²，王明荣¹，张钰^1，*

中国医学科学院肿瘤医院肿瘤研究所，分子肿瘤学国家重点实验室，北京 100021

收稿日期:2014-04-01 修回日期:2014-08-15 出版日期:2014-09-30 发布日期:2014-09-30
通讯作者: 张钰，E-mail：zhangyu909@126.com
作者简介:曹迎亚（1990- ），女，安徽芜湖人，硕士研究生，研究方向：麻醉与应激。
基金资助:
国家自然科学基金重点项目（81330052），国家自然科学基金-创新研究群体科学基金(81321091)，国家高技术研究发展规划(863计划)项目(2012AA02A503)

Establishment of an esophageal cell line with stable expression of inducible PLK1-shRNA

CAO Ying-ya^1，2，CHE Yi-qun ¹，CHEN Qun¹ ，MA Wen-tao¹ ，LIU Yang ¹，HAO Jia-jie¹，CAI Yan¹，LU Wei-hua²，WANG Ming-rong¹，ZHANG Yu^1，*

State Key Laboratory of Molecular Oncology, Cancer Institute of Cancer Hospital, Chinese Academy of Medical Sciences, Beijing 100021

Received:2014-04-01 Revised:2014-08-15 Online:2014-09-30 Published:2014-09-30

摘要/Abstract

摘要：

目的：建立可条件性诱导PLK1-shRNA稳定表达的食管癌细胞株，为深入研究PLK1在食管癌发生发展中的作用及分子机制提供细胞模型。方法：合成PLK1-shRNA寡核苷酸，退火后连接到慢病毒表达载体pLKO-Tet-On并转化至感受态细胞Stbl3，利用菌落PCR和测序分析鉴定阳性重组子。用pLKO-shPLK1-Tet-On重组质粒和包装质粒共转染293T细胞，收集病毒上清，感染食管癌细胞KYSE510，利用嘌呤霉素筛选可诱导PLK1-shRNA稳定表达的细胞株。采用qRT-PCR和Western blot检测强力霉素(Dox)诱导PLK1-shRNA表达的效率。结果：菌落PCR和测序分析结果显示，pLKO-shPLK1-Tet-On重组质粒中PLK1-shRNA的序列及插入位点正确。包装、收获病毒并感染KYSE510细胞后，筛选获得了具有嘌呤霉素抗性的稳定细胞株KYSE510-shPLK1-Tet-On。qRT-PCR和Western blot的检测结果显示，0.1 μg/mL Dox即可显著下调KYSE510-shPLK1-Tet-On细胞中PLK1的表达。结论：成功构建了诱导型PLK1-shRNA慢病毒表达载体，并筛选获得了可条件性诱导PLK1稳定敲降的食管癌细胞株，为进一步探讨PLK1异常表达与食管癌发生发展的关系提供了理想的细胞模型。

关键词: PLK1, shRNA, 诱导表达, 慢病毒载体, 食管癌

Abstract:

OBJECTIVE: The aim of the study was to establish a suitable cell model for investigating the role and mechanism of PLK1 overexpression in esophageal cancer through inactivation of PLK1 expression employing lentiviral-mediated inducible shRNA expression system. METHODS：Chemically synthesized PLK1-shRNA oligonucleotides were annealed and ligated into the lentiviral vector pLKO-Tet-On. The ligation products were transformed into the competent E. coli Stbl3 cells. Colony PCR and sequencing analysis were used to identify the positive recombinants. The pLKO-shPLK1-Tet-On construct and packaging plasmids were co-transfected into 293T cells to produce the lentiviral particles. Esophageal cancer cells KYSE510 were infected with the viral supernatant and the stable cell strain was selected with puromycin. Doxcyclin-induced expression efficiency of PLK1-shRNA was determined by qRT-PCR and Western blotting. RESULTS：Colony PCR and sequencing analysis showed that PLK1-shRNA oligos were correctly inserted into the pLKO-Tet-On vector. The stable cell strain KYSE510-shPLK1-Tet-On was obtained through infecting the lentivirus expressing inducible PLK1-shRNA and then selected with puromycin. The results of qRT-PCR and Western blotting indicated that the expression of PLK1 in KYSE510-shPLK1-Tet-On cells could be markedly downregulated by 0.1 μg/mL Dox. CONCLUSION：We successfully constructed a lentivirus-based inducible PLK1-shRNA expression vector and established an esophageal cancer cell line with stable expression of inducible PLK1-shRNA，providing an ideal cell model for further exploring the relationship between aberrant PLK1 expression and development and progression of esophageal cancer.

Key words: PLK1, shRNA, inducible expression, lentivirus expression vector, esophageal cancer

曹迎亚，车轶群，陈群，马文韬，刘洋，郝佳洁，蔡岩，鲁卫华，王明荣，张钰. 可条件性诱导PLK1稳定敲降的食管癌细胞株的建立[J]. 癌变·畸变·突变, doi: 10.3969/j.issn.1004-616x.2014.05.006.

CAO Ying-ya，CHE Yi-qun，CHEN Qun，MA Wen-tao，LIU Yang，HAO Jia-jie，CAI Yan，LU Wei-hua，WANG Ming-rong，ZHANG Yu. Establishment of an esophageal cell line with stable expression of inducible PLK1-shRNA[J]. Carcinogenesis, Teratogenesis & Mutagenesis, doi: 10.3969/j.issn.1004-616x.2014.05.006.

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可条件性诱导PLK1稳定敲降的食管癌细胞株的建立

Establishment of an esophageal cell line with stable expression of inducible PLK1-shRNA

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