Construction of a lentiviral vector of FoxM1 shRNA and its transfection into human prostate cancer cell lines in vitro
- VernacularTitle:FoxM1 shRNA慢病毒载体构建及感染人前列腺癌细胞的稳定细胞株筛选
- Author:
Yiru WANG
1
;
Binwei YAO
;
Yan ZHANG
;
Mingbo ZHANG
;
Hanjing GAO
;
Jie TANG
Author Information
1. 解放军总医院超声科
- Keywords:
FoxM1;
prostate cancer;
lentiviral vector;
stable cell line
- From:
Journal of Southern Medical University
2015;(9):1227-1233
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a recombinant lentiviral vector that co-express green fluorescent protein (GFP) and FoxM1 shRNA and establish a prostate cancer cell line with stable FoxM1 down-regulation. Methods Three interfering sequences targeting FoxM1 were designed and inserted into the lentiviral vector pHBLV-U6-ZsGreen-Puro. After identification by DNA sequencing, the lentiviral vectors carrying Foxm1 shRNA were packaged in 293 cells. The lentiviral particles were collected to infect human prostate cancer DU-145 cells, and the transfection efficiency was observed under fluorescence microscope; the interference efficiency was assessed using real-time PCR. DU-145 cells with stable FoxM1 down-regulation were screened with puromycin, and the expression level of FoxM1 was detected by Western blotting and the cell growth was observed using MTT assay. The stably transfected cells were examined for cell apoptosis and cell clone formation capacity with flow cytometry and colony formation assay. Results DNA sequencing demonstrated successful construction of the 3 FoxM1 shRNA lentivirus vectors. Real-time PCR showed a high interference efficiency of FoxM1 shRNA1 vector, which resulted in obvious down-regulation of FoxM1 in DU-145 cells. Western blotting showed that the expression of FoxM1 protein was decreased in FoxM1 shRNA1 lentivirus-transfected cells, which displayed a suppressed cell proliferation, increased apoptosis rate, and attenuated clonogenic ability. Conclusion We have successfully established a prostate cancer cell model with stable FoxM1 down-regulation, which shows lowered proliferative and clonogenic activities with increased cell apoptosis.
