Construction of cell line of small hairpin RNA-mediated inhibition of HER2/neu gene expression.
- Author:
Juan FU
1
;
Yi-guo JIANG
;
Ying JU
;
Yue-lan SHEN
;
Xue-min CHEN
Author Information
- Publication Type:Journal Article
- MeSH: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; toxicity; Base Sequence; Cell Line, Transformed; Epithelial Cells; drug effects; metabolism; Gene Expression; Genes, erbB-2; Humans; Molecular Sequence Data; RNA, Small Interfering; Reverse Transcriptase Polymerase Chain Reaction; Transfection
- From: Chinese Journal of Preventive Medicine 2009;43(8):714-717
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo 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).
METHODSThe 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.
RESULTSThe 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.
CONCLUSIONPlasmid-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.
