[Effective inhibition of hTERT expression by RNA interference on the radiosensitivity of human laryngeal cancer Hep-2 cell line].
OBJECTIVE
To construct an eukaryotic expression vector of human telomerase reverse transcriptase (hTERT) gene specific shRNA, and investigate the effect of pshRNA-hTERT combined with gamma-irradiation on cell survival and telomerase activity.


METHODS
According to the coding sequence of hTERT mRNA, the target of RNAi was designed, and recombinant expression plasmid pshRNA-hTERT was constructed. The vector was transfected into Hep-2 cells. The radiosensitivity of Hep-2 cells was determined by clonogenic assay. Telomeric repeat amplification protocol (TRAP-PCR-ELISA) was used to observe the telomerase activity in each group. Results Recombinant expression vector pshRNA-hTERT was successfully transfected into Hep-2 cells. The hTERT expression inhibition rate reached 60. 8%. pshRNA-hTERT not only inhibited telomerase activity of Hep-2, but also inhibited the raise of telomerase activity induced by gamma-irradiation. Exposure of Hep-2 cells to pshRNA-hTERT for 24 hrs before irradiation resulted in a decrease in mean surviving fraction of Hep-2 cells compared with cells of group with irradiation alone (67. 7% vs 85. 7%, P <0. 05) .


CONCLUSIONS
RNAi showed a significant inhibitory effect to the expression of hTERT. The results indicate that pshRNA-hTERT can effectively inhibit telomerase activity of Hep-2 cells treated or untreated with 2 Gy gamma-irradiation and significantly enhance the radiosensitivity of Hep-2 cells in vitro. The role of radiosensitization of pshRNA-hTERT may be related with the inhibition of telomerase activity.
Qiu
Hui-bing
HB
Department of Oncological Chemotherapy and Radiotherpy, Zhongnan Hospital, Wuhan University, Wuhan 430071, China.
Zhou
Yun-feng
YF
Zhou
Fu-xiang
FX
Xie
Cong-hua
CH
Luo
Zhi-guo
ZG
Yu
Hai-jun
HJ
Liu
Shi-quan
SQ
D002294:Carcinoma, Squamous Cell / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics / Q000528:radiation effects; D003037:Cobalt Radioisotopes; D004797:Enzyme-Linked Immunosorbent Assay; D005720:Gamma Rays; D006801:Humans; D007822:Laryngeal Neoplasms / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D010957:Plasmids; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D003037:Cobalt Radioisotopes; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D019098:Telomerase