Abstract: Objective To investigate the effect of small interfering RNA(siRNA)directed against human telomerase reverse transcriptase(hTERT)on the telomerase activity and proliferation of cutaneous T-cell lymphoma(CTCL)cell line Hut78.Methods Two pairs of siRNAs synthesized with T7 RNA polymerase in vitro transcription were transfected into Hut78 cells by calcium phosphate co-precipitation. The hTERT mRNA expression and telomerase activity were testedby reverse transcription polymerase chain reaction(RT-PCR)and telomeric repeat amplification protocol(TRAP),respectively.MTT assay was performed to evaluate the cell proliferation.Apoptosis and cell cycles were assessed by using flow cytometry.Results High production of siRNAs were synthesized with T7 RNA polymerase in vitro transcription.The two pairs of siRNAs(250 ng)could significantly down-regulate the mRNA expression of hTERT by about 65%,and they suppressed telomerase activity by 68% and 64%,respectively,at 48 h after the transfection. siRNAs could inhibit cell proliferation in a dose-dependent manner;most pronounced inhibition was observed at a dose of 1000 ng.The apoptosis rates of Hut78 cells were 15.86% and 11.10% respectively after transfected with the two pairs of siRNA.No significant changes were found for the above parametres either in calcium phosphate group or in the blank control group.Conclusions The hTERT-siRNA synthesized with T7 RNA polymerase in vitro transcription could suppress the telomerase activity of Hut78 cells,down-regulate the expression of hTERT gene in these cells,inhibit the proliferation,and induce the apoptosis of these cells.

Key words: Apoptosis, Telomerase, Tumor cell, cultured, Lymphoma, small interfering, RNA