[Construction of cell line of small hairpin RNA-mediated inhibition of HER2/neu gene expression].
OBJECTIVE
To establish the stable inhibition of HER2/neu expression by vector-mediated small hairpin RNA in malignant transformed human bronchial epithelial cell line induced by anti-benzo(a)pyrene-trans-7, 8-dihydrodiol-9, 10-epoxide (anti-BPDE).


METHODS
The pSIREN-RetroQ-neu recombinant vector targeting HER2/neu was constructed and confirmed by restriction and sequencing analysis, then it was transfected into anti-BPDE malignant transformed 16HBE cells (16HBE-T) through lipofectamine 2000. The control groups included the 16HBE-T cells transfected with negative control vector (negative control) and 16HBE-T. The cells transfected with vectors were screened by puromycin. The HER2/neu mRNA and protein expressions in the vector-transfected 16HBE-T cells were detected by RT-PCR and Western blot method respectively.


RESULTS
The pSIREN-RetroQ-neu recombinant vector which inhibited HER2/neu mRNA and protein expressions in the 16HBE-T was constructed. The level of HER2/neu mRNA in the 16HBE-T cells transfected with pSIREN-RetroQ-neu was significantly reduced as compared to the negative control and blank control cells (0.114 +/- 0.003 vs.blank control 0.186 +/- 0.001, t = 39.154, P < 0.05; and negative control 0.182 +/- 0.015, t = 7.564, P < 0.05), while its level did not differ significantly between negative control cells and blank control of 16HBE-T (t = -0.409, P > 0.05). HER2/neu protein level in pSIREN-RetroQ-neu transfected cells was inhibited by 40% and 39% respectively.


CONCLUSIONS
Plasmid-based shRNA expression systems targeted against HER2/neu gene were generated successfully, which resulted in down-regulation of HER2/neu gene expression in the 16HBE-T efficiently.
Fu
Juan
J
Department of Occupational and Environmental Health and MOE Key Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Jiang
Yi-guo
YG
Ju
Ying
Y
Shen
Yue-lan
YL
Chen
Xue-min
XM
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000633:toxicity*; D001483:Base Sequence; D002461:Cell Line, Transformed; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D015870:Gene Expression; D018734:Genes, erbB-2; D006801:Humans; D008969:Molecular Sequence Data; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D034741:RNA, Small Interfering; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide