Synthesis of hTERT-siRNA and hTR-siRNA by in vitro Transcription and Their Inhibition on Telomerase Activity in Hut78 Cells / 中华皮肤科杂志

ABSTRACT

Objectives To synthesize human telomerase reverse transcriptase (hTERT)- and human tolemerase RNA (hTR)- small interfering RNA (siRNA) and investigate their effects on telomerase activity in the cutaneous T-cell lymphoma (CTCL) cell line Hut78. Methods Two types of hTERT- and hTR- siRNAs were synthesized with T7 RNA polymerase via in vitro transcription, then either mixed with Hut78 cell lysates directly or transfected into Hut78 cells by calcium phosphate co-precipitation. Telomerase activity was tested by telomeric repeat amplification and polyacrylamide gel electrophoresis. Results With T7 RNA polymerase, hTERT- and hTR- siRNAs were synthesized efficiently with a concentration of 22.4?g siRNA per 40?L siRNA reaction mix. Telomerase activity was suppressed significantly by either of the siRNAs. The inhibition rate was 87% in the cell lysate group treated with siRNA directly, and 75% in the cell group Iransfected with siRNA. Conclusions The in vitro transcription of siRNA with T7 RNA polymerase is technically simple, costeffective, and can produce siRNA in an efficient way. hTERT- and hTR-siRNA can down-regulate telomerase activity significantly in Hut78 cells.