[Construction and selection of siRNA expression cassettes targeting human telomerase reverse transcriptase gene in vitro].
OBJECTIVE
To 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.


METHODS
Four 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.


RESULTS
The 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.


CONCLUSIONS
Specific 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.
Liu
Xi-qiang
XQ
Department of Oral and Maxillofacial Surgery, Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou 510055, China.
Huang
Hong-zhang
HZ
Pan
Chao-bin
CB
Wang
Fang-jin
FJ
Zhang
Bin
B
Liang
Wei-wen
WW
D002294:Carcinoma, Squamous Cell / Q000201:enzymology* / Q000235:genetics; D045744:Cell Line, Tumor; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D034741:RNA, Small Interfering / Q000235:genetics*; D019098:Telomerase / Q000235:genetics*; D014062:Tongue Neoplasms / Q000201:enzymology* / Q000235:genetics; D014162:Transfection
D034741:RNA, Small Interfering; C509186:TERT protein, human; D019098:Telomerase