[Specific inhibition of hTERT gene expression by short interfering RNAs in gastric cancer SGC7901 cell].
OBJECTIVE
Activation of hTERT, the human telomerase catalytic subunit, has been implicated as the critical event in triggering telomerase activity of cancer cells. In present research, we investigated whether RNA interference (RNAi) induced by small interference RNA (siRNA) could suppress human telomerase catalytic unit (hTERT) gene expression in gastric SGC7901 cells.


METHODS
As a pilot study, we utilized green fluorescent protein (GFP) plasmid pCX-GFP (5 510 bp) as a reporter system and generated constructs SHi-pU6-GFP expressing small hairpin RNA (shRNA) specific for green fluorescence protein (GFP) in K562 and SGC7901 cell respectively. Furthermore, we constructed pU6-hTERT-siRNAs carried hairpin siRNA for hTERT gene and transfected in SGC7901 by using Lipofectamine trade mark 2000. The expression of hTERT gene was detected by reverse transcription polymerase chain reaction (RT-PCR) and fluorescence quantitative polymerase chain reaction (FQ-PCR) assay.


RESULTS
Our pilot study showed the short hairpin RNA (shRNA) expression vector driven by the murine U6 small nuclear RNA promoter can specifically induce potent gene knockdown effect (i.e., inhibit GFP expression specifically) when transfected transiently into SGC7901 cell. The constructed pU6-hTERT-siRNAs carried hairpin siRNA for hTERT gene was proved to be the same as designed by restriction endonuclease analysis. pU6-hTERT-siRNAs were successfully transferred into SGC7901 cell and their stable expression were obtained. The expression of hTERT gene were specific inhibited by pU6-hTERT-siRNAs in SGC7901 cell.


CONCLUSIONS
Short hairpin RNAs (shRNAs) could induce sequence-specific hTERT gene silencing in SGC7901 cell. Our results prove the feasibility of the U6 promoter-driven shRNA expression technique to be used to cancer gene therapy.
Ma
Jin-Ping
JP
Department of General Surgery, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China.
Zhan
Wen-Hua
WH
Wang
Jian-Ping
JP
Peng
Jun-Sheng
JS
Gao
Jin-Song
JS
Yin
Qin-Wei
QW
D020134:Catalytic Domain / Q000235:genetics*; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D010957:Plasmids; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D012342:RNA, Small Nuclear / Q000235:genetics; D011995:Recombination, Genetic; D013274:Stomach Neoplasms / Q000235:genetics* / Q000473:pathology; D019098:Telomerase / Q000235:genetics*; D014162:Transfection
D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear; C405486:U6 small nuclear RNA; D049452:Green Fluorescent Proteins; D019098:Telomerase