[Inhibition of telomerase activity by ribozyme targeted to human telomerase transcriptase]

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.