RNA干扰抑制Hep-2细胞人端粒酶逆转录酶基因表达对放射敏感性的影响

Effective inhibition of hTERT expression by RNA interference on the radioseusitivity of human laryngeal cancer Hep-2 cell line

目的构建人端粒酶逆转录酶（hTERT）基因特异性短发夹样RNA（shRNA）真核表达载体，观察其联合射线对人喉癌Hep-2细胞端粒酶活性和细胞存活的影响，研究hTERT基因在放射增敏中的作用。方法根据hTERT mRNA编码序列设计RNA干扰（RNAi）靶点，构建重组表达质粒pshRNA-hTERT，构建成功后，转染Hep-2细胞并作射线处理，用端粒重复序列扩增方法（TRAP-PCR-ELISA）检测端粒酶活性的动态变化，采用克隆形成分析方法观察质粒pshRNA—hTERT对Hep-2细胞放射敏感性的影响，计算放射增敏比SERSF2。结果转染质粒pshRNA-hTERT后，Hep-2细胞的hTERT mRNA表达抑制率为60．8％，质粒pshRNA-hTERT不仅能够抑制Hep-2细胞的端粒酶活性（P〈0．05），而且还可以抑制辐射诱导的端粒酶活性上升（P〈0．05）。照射前经pshRNA-hTERT处理24h，明显降低了2Gy照射后Hep-2细胞的存活分数（85．7％），与单纯放射组（67．7％）相比，差异有统计学意义（P〈0．05），pshRNA-hTERT的放射增敏比SERSF2为1．27。结论RNAi显著抑制了靶基因hTERT的表达，质粒pshRNA-hTERT能明显抑制2Gy照射诱导的Hep-2细胞端粒酶活性升高，并显著提高其体外放射敏感性；质粒pshRNA-hTERT的放射增敏作用可能与端粒酶活性受抑制有关。

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 γ-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 γ- 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） .Conclusion 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 γ-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.