Effect of shRNA inhibiting hTERT gene expression combined with γ-irradiation on human laryngeal cancer cells

Objective: To construct an eukaryotic expression vector of human telomerase reverso transcriptase (hTERT) gene specific shRNA, and investigate the effect of pshRNA-hTERT combined with γ-irradiation on telomerase activity and DNA damage. Methods: The recombinant expression plasmid pshRNA-hTERT was constructed and transfected into Hep-2 cells. The telomerase activity was examined by the PCR-hased telomeric repeat amplification protocol (TRAP). DNA single-stranded break (SSB) and the DNA double-stranded break (DSB) were detected by Comet assay. The xenograft model of human laryngeal carcinoma with the same genetic background and different radiosensitivity (Hep-2 and Hep-2R) was established in nude mice. The mixture of pshRNA-hTERT and liposome was injected to the transplanted tumor to observe the inhibition of the tumor growth. The cell apoptosis was detected by TUNEL. The hTERT protein expression was determined by streptavidin-peroxidase conjugated method (AP). Results: Recombinant expression plasmid pshRNA-hTERT was successfully constructed and transfected into Hep-2 cells. The hTERT expression inhibition rate reached 60.78 %. pshRNA-hTERT not only inhibited telomerase activity of Hep-2 inehiding the increase of telomerase activity induced by γ-irradiation, but also inhibited the repair of the SSB and DSB induced by irradiation in the human laryngeal carcinoma xenograft in nude mice with the same genetic background and different radiosensitivity. The pshRNA-hTERT combined with γ-irradiation could inhibit the growth of transplanted tumor (Hep-2: EPO = 1.79; Hep-2R: EPO = 2.01) with reduced telomerase activity and hTERT protein expression. Conclusions: The eukaryotic expression vector pshRNA-hTERT could enhance the radiosensitivity of Hep-2 cells in vitro and the human laryngeal carcinoma xenograft in nude mice which had the same genetic background with different radiosensitivity. (authors)