Construction of pVAX-WIF-1 Eukaryotic Expression Vector and Its Anti-tumor Effect on Lung Cancer
10.3779/j.issn.1009-3419.2015.07.04
- VernacularTitle:pVAX-WIF-1真核表达载体的构建及其抗肺癌作用
- Author:
AN NING
1
;
LUO XINMEI
;
YE SUJUAN
;
WANG YU
;
YANG WEIHAN
;
JIANG QIANQIAN
;
ZHU WEN
Author Information
1. 610072成都，四川省医学科学院，四川省人民医院肿瘤科
- Keywords:
Lung cancer;
WIF-1;
A549;
Gene therapy;
Eukaryotic expression vector
- From:
Chinese Journal of Lung Cancer
2015;(7):409-415
- CountryChina
- Language:Chinese
-
Abstract:
Background and objectiveWIF-1 is an important tumor-suppressing gene in lung cancer, and its encoding protein WIF-1 can reduce proliferation and promote apoptosis by inhibiting Wnt/β-catenin signaling in lung can-cer. hTis study constructs a eukaryotic expression plasmid carrying WIF-1 using FDA-approved clinical plasmid pVAX and explores the anti-tumor effect of pVAX-WIF-1 on A549 lung cancer cellsin vitro andvivo.MethodshTe DNA fragment of human WIF-1 coding sequence was ampliifed by PCR and was cloned into the multiple cloning sites of eukaryotic expression vector pVAX to construct pVAX-WIF-1. A recombinant plasmid was transfected into lung cancer A549 cells, and the expression ofWIF-1 genes was veriifed by Western blot atfer transfection. Subsequently, the effect of pVAX-WIF-1 on cell apoptosis and proliferation was identiifed by MTT assay, staining A549 cells with Hoechst 3235, and lfow cytometry. Finally, the A549 sub-cutaneous xenogratf was established to detect the effect of pVAX-WIF-1 on lung tumor growthin vivo.Results hTe results of restriction enzyme digestion, PCR, and sequencing indicated that eukaryotic expression plasmid pVAX-WIF-1 was successfully constructed. hTe protein expression level of WIF-1 was increased in the transfected A549 cells. Further results showed that transfection with pVAX-WIF-1 signiifcantly inhibited proliferation and promoted apoptosis in A549 cells. Moreover, pVAX-WIF-1 signiifcantly inhibited the tumor growth of the A549 subcutaneous xenogratfin vivo.ConclusionhTe recombinant eukaryotic expression vector pVAX-WIF-1 was successfully constructed. Transfection with pVAX-WIF-1 could signiifcantly inhibit proliferation and promote apoptosis of lung cancer A549 cells and also effectively inhibit the tumor growth of the A549 subcutaneous xenogratfin vivo. Our research can contribute to clinical applications of WIF-1 in lung cancer gene therapy.
