Inhibition of human telomerase reverse transcriptase in hep-2 cells using short hairpin RNA expression vectors.
OBJECTIVE
Telomerase activity is mainly regulated by the human telomerase reverse transcriptase (hTERT) gene. Our objective was to investigate the effect of short hairpin RNA (shRNA) on hTERT expression and telomerase activity in laryngeal cancer cells.


METHODS
Short hairpin RNA expression vectors targeting the messenger RNA of hTERT were constructed. Cells were treated with shRNA expression vectors directed against 2 different hTERT sites, control vectors that included mismatched shRNA and those without shRNA. The expression of hTERT was determined by reverse-transcriptase polymerase chain reaction and Western blotting. The activity of telomerase was measured by telomeric repeated amplification enzyme-linked immunosorbent assay. The cell viability was examined using the 3-(4,5-dimethyl thizol-2-yl) 2,5-diphenyl tetrazolium bromide assay.


RESULTS
We found that treatment of shRNA expression vectors induced a significant decrease in hTERT messenger RNA expression, the level of hTERT protein, telomerase activity, and cell viability. All of these effects were seen regardless of the target site, and the shRNA control showed none of these effects.


CONCLUSIONS
Our results suggest that shRNA directed against hTERT inhibits telomerase activity through suppression of the hTERT expression in laryngeal cancer cells and that RNA interfering technology may be a promising strategy for the treatment of laryngeal cancers.
Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan University, Wuhan, China. shimingchen0468@hotmail.com
Shi-Ming
SM
Chen
Ze-Zhang
ZZ
Tao
Qing-Quan
QQ
Hua
Dan
D
Liu
Hua-Ming
HM
Chi
Qing
Q
Cai
D017209:Apoptosis; D015153:Blotting, Western; D020134:Catalytic Domain / Q000187:drug effects* / Q000235:genetics; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D007822:Laryngeal Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D010957:Plasmids / Q000494:pharmacology*; D016337:RNA, Catalytic / Q000235:genetics / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics*; D012334:RNA, Neoplasm / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019098:Telomerase / Q000037:antagonists & inhibitors* / Q000235:genetics
D004268:DNA-Binding Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012334:RNA, Neoplasm; C418481:hairpin ribozyme; D019098:Telomerase