癌变·畸变·突变Issue(5)：348-352,5.DOI:10.3969/j.issn.1004-616x.2014.05.006
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
摘要
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 competentE. 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 0.1μg/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.关键词
PLK1/shRNA/诱导表达/慢病毒载体/食管癌Key words
PLK1/shRNA/inducible expression/lentivirus expression vector/esophageal cancer分类
生物科学引用本文复制引用
曹迎亚,张钰,车轶群,陈群,马文韬,刘洋,郝佳洁,蔡岩,鲁卫华,王明荣..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,2014,(5):348-352,5.基金项目
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) （81321091）
