Suppression of telomerase activity by plasmid-mediated RNA interference.
- Author:
Yan LI
1
;
Mingyuan LI
;
Ying PENG
;
Zhonghua JIANG
;
Wanyi LI
;
Hong LI
Author Information
1. Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
- Publication Type:Journal Article
- MeSH:
Base Sequence;
Gene Expression Regulation, Enzymologic;
Gene Expression Regulation, Neoplastic;
HeLa Cells;
Humans;
Molecular Sequence Data;
Plasmids;
genetics;
RNA, Small Interfering;
genetics;
pharmacology;
Telomerase;
antagonists & inhibitors;
genetics;
Transfection
- From:
Journal of Biomedical Engineering
2006;23(3):615-619
- CountryChina
- Language:Chinese
-
Abstract:
This study was aimed to construct a plasmid expressing siRNA specific for the human telomerase reverse transcriptase (hTERT) gene and to evaluate the ability of small interference RNA(siRNA) for inhibiting telomerase activity in HeLa cells. 64 nucleotides, in which 19 nt were homologous with hTERT gene, were chemically synthesized, annealed and linked into pSUPER to get pSUP-hTE. Then pSUP-hTE was digested with enzyme. We obtained its fragmant concluding promoter and 64nt. So we cloned it into pEGFP-C1 for constructing pEGFP-hTE which contains neo gene and the enhanced green fluorescent protein (EGFP). Recombinant pEGFP-hTE was transfected to HeLa cells. These cells were screened with medium containing G418. When stable colonies appeared, G418-resistant cells were harvested and propagated. At the different cell generations, hTERT mRNA and protein expression, telomerase activity and cell growth activity were analyzed. Compared with control cells, HeLa cells transfected with pEGFP-hTE showed that hTERT mRNA level and hTERT protein expression decreased and telomerase activity reduced by 38%, but the cells growth activity displayed no changes. So pEGFP-hTE could specifically inhibit expression of hTERT and telomerase activity. These results suggested that siRNA targeting hTERT gene might provide a new strategy for cancer biotherapy.
