1. Inhibition of Human Telomerase Reverse Transcriptase in Hep-2 Cells Using Short Hairpin RNA Expression Vectors
- Author
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Shi-Ming Chen, Qing-Quan Hua, Qing Cai, Ze-Zhang Tao, Dan Liu, and Hua-Ming Chi
- Subjects
Telomerase ,Blotting, Western ,Genetic Vectors ,Apoptosis ,Enzyme-Linked Immunosorbent Assay ,In Vitro Techniques ,Biology ,Small hairpin RNA ,Catalytic Domain ,Cell Line, Tumor ,Humans ,RNA, Catalytic ,Telomerase reverse transcriptase ,RNA, Messenger ,RNA, Neoplasm ,Viability assay ,Laryngeal Neoplasms ,neoplasms ,Messenger RNA ,Expression vector ,Reverse Transcriptase Polymerase Chain Reaction ,RNA ,General Medicine ,Transfection ,Molecular biology ,DNA-Binding Proteins ,enzymes and coenzymes (carbohydrates) ,Otorhinolaryngology ,embryonic structures ,Surgery ,Plasmids - Abstract
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. Design 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. Conclusion 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.
- Published
- 2006