| HU Liu,ZHOU Fu-xiang,LEI Han,et al.Effect of shRNA inhibiting hTERT gene expression combined with γ-irradiation on human laryngeal cancer cells[J].Chinese Journal of Radiological Medicine and Protection,2009,29(3):253-258 |
| Effect of shRNA inhibiting hTERT gene expression combined with γ-irradiation on human laryngeal cancer cells |
| Received:July 23, 2008 |
| DOI：10.3760/cma.j.issn.0254-5098.2009.03.002 |
| KeyWords:pshRNA-hTERT Radiosensitivity DNA damage repair Laryngeal cancer xenograft Nude mice |
| FundProject:国家自然科学基金(30171063) |
| Author Name | Affiliation | E-mail | | HU Liu | Department of Chemoradiotherapy, Zhongnan Hospital of Wuhan University, Wuhan 430071, China | | | ZHOU Fu-xiang | Department of Chemoradiotherapy, Zhongnan Hospital of Wuhan University, Wuhan 430071, China | happyzhoufx@sina.com | | LEI Han | Department of Chemoradiotherapy, Zhongnan Hospital of Wuhan University, Wuhan 430071, China | | | ZHANG Xi-mei | Department of Chemoradiotherapy, Zhongnan Hospital of Wuhan University, Wuhan 430071, China | | | QIU Hui-bing | Department of Chemoradiotherapy, Zhongnan Hospital of Wuhan University, Wuhan 430071, China | | | DAI Jing | Department of Chemoradiotherapy, Zhongnan Hospital of Wuhan University, Wuhan 430071, China | | | HUANG Cheng-hu | Department of Chemoradiotherapy, Zhongnan Hospital of Wuhan University, Wuhan 430071, China | | | XIE Cong-hua | Department of Chemoradiotherapy, Zhongnan Hospital of Wuhan University, Wuhan 430071, China | | | LIU Shi-quan | Department of Chemoradiotherapy, Zhongnan Hospital of Wuhan University, Wuhan 430071, China | | | ZHOU Yun_feng | Department of Chemoradiotherapy, Zhongnan Hospital of Wuhan University, Wuhan 430071, China | |
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| Abstract:: |
| 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 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-based 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 including 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. |
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