Long-term suppression of telomerase expression in HeLa cell clones, transfected with an expression vector carrying siRNA targeting hTERT mRNA.
HeLa cell cultures were used as model systems for small interfering RNA (siRNA) induced knockdown of mRNA expression of the human telomerase catalytic subunit, telomerase reverse transcriptase (hTERT). Four 21-bp siRNAs targeting different sites of the hTERT mRNA were designed, and the siRNA molecules produced by a T7 transcription system in vitro. In transient transfection assays on HeLa cells, only one of the tested siRNAs produced a potent knockdown effect on hTERT mRNA expression, associated with the suppression of telomerase activity (both reduced by approximately 50%). An expression vector encoding a hairpin siRNA against the effective hTERT mRNA target site was generated, and HeLa clones stably expressing hTERT-specific siRNA were created. Two clones showed extremely reduced hTERT mRNA expression, associated with unusually short telomeres, the inhibition of cell growth and the induction of senescence and apoptosis. Thus, there was obvious loss of viability in cells lacking hTERT expression and carrying short telomeres. This was most prominent in the clone that showed prolonged reductions (over two months) in both hTERT expression and telomerase activity. Thus, our data clearly show that long-term suppression of telomerase expression by siRNA is an attainable goal, at least in a HeLa cell model system.
Kurvinen
Kaisa
K
Department of Oral Pathology and Oral Radiology, Institute of Dentistry and MediCity Research Laboratory, Faculty of Medicine, University of Turku, Turku, Finland.
Syrjänen
Stina
S
Johansson
Bo
B
D017209:Apoptosis; D049109:Cell Proliferation; D048430:Cell Shape; D002999:Clone Cells; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D034622:RNA Interference; D012333:RNA, Messenger; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D002583:Uterine Cervical Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology
D012333:RNA, Messenger; D034741:RNA, Small Interfering; C509186:TERT protein, human; D019098:Telomerase