Construction and selection of siRNA expression cassettes targeting human telomerase reverse transcriptase gene in vitro.
- Author:
Xi-qiang LIU
1
;
Hong-zhang HUANG
;
Chao-bin PAN
;
Fang-jin WANG
;
Bin ZHANG
;
Wei-wen LIANG
Author Information
- Publication Type:Journal Article
- MeSH: Carcinoma, Squamous Cell; enzymology; genetics; Cell Line, Tumor; Gene Expression; Genetic Vectors; Humans; RNA, Small Interfering; genetics; Telomerase; genetics; Tongue Neoplasms; enzymology; genetics; Transfection
- From: Chinese Journal of Stomatology 2006;41(7):403-406
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo determine whether the human telomerase reverse transcriptase (hTERT) gene silencing could be effectively induced by PCR-derived siRNA expression cassettes (SEC) transfected by the fifth generation polyamidoamine dendrimer (G5 PAMAM-D) in Tca8113 cells.
METHODSFour SEC were rationally designed and constructed based on a two-step PCR reaction. The SEC were then transferred into Tca8113 cells using G5 PAMAM-D, and hTERT expression was investigated by real-time fluorescence-quantitative reverse transcriptase-PCR and western blot analysis.
RESULTSThe RNA interference effects of the SEC targeted for varying hTERT mRNA positions showed a significant disparity. Among them, SEC-A revealed the most potent inhibitory effects (above 95% of reduction), followed by SEC-D and SEC-C, and SEC-B had no effect on hTERT expression (P > 0.05). That the endogenous hTERT gene silencing induced by G5 PAMAM dendrimer-mediated SEC-A was highly sequence-specific, and multiple transfection as well as properties of the vectors were routinely attributable to the specific suppression.
CONCLUSIONSSpecific inhibition of endogenous hTERT expression by use of a PCR-based short hairpin siRNA technique and dendrimer transfer system may serve as a novel strategy for treatment of tongue cancers expressing hTERT in vitro.