Inhibition of
Telomerase Activity by Ribozyme Targeted to Human Telomerase Transcriptase
QU Yi,LIU Shu-Qiu,PENG Wen-Zhen,LIU
Bai-Lin*
( Laboratory of Medical Molecular Biology,Sichuan University,Chengdu 610041,China
)
Abstract Human
telomerase reverse transcriptase (hTERT) is the catalytic subunit and the key
factor which controls the telomerase activity,so it is the best choice to
inhibit telomerase through controling hTERT expression.In this work,a hammer
head ribozyme directed against the hTERT mRNA (hTERTRZ) was designed and
synthesized to serve as a telomerase inhibitor.In order to test its in vitro
cleavage activity,two in vitro transcription plasmids containing hTERTRZ
and hTERT gene respectively were constructed. Ribozyme RNA and
DIG-labeled-hTERT were synthesized by in vitro transcription.In vitro
cleavage reactions were carried out by mixing the hTERTRZ with
DIG-labeled-hTERT under different reaction conditions,and cleavage bands were
detected by digoxin chemiluminescent assay. hTERTRZ showed a specific cleavage
activity against the hTERT used as template. To investigate its in vivo
effect of telomerase inhibition in tumor cells,a eukaryotic expression plasmid
containing the hTERT ribozyme gene was introduced into HeLa cells and hepatoma
cells by using LipofectAMINE.In the transfectants,the level of intact hTERT
mRNA and the telomerase activity were clearly reduced,and the telomere length
of these clones was apparently shortened at the beginning period,then kept a
fixed value without further shortening.All the transfectants with ribozyme grew
clearly more slowly than the parental cell line.The doubling time of the
tansfectants prolonged compared to the negative control,but no apparent
apoptosis was shown even at their 37th passage.These findings suggest the
potential application of this ribozyme as a new theraputic agent directed
against immortalized cancer cells.
Key words ribozyme;telomerase;human telomerase reverse
transcriptase;tumor
*Corresponding author:
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