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

Abstract

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

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

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