Construction of a novel vector expressing the fusion suicide gene yCDglyTK and hTERT-shRNA and its antitumor effects

Li,Jia; Zhang,Guiying; Liu,Ting; Gu,Huan; Yan,Lu; Chen,Bolin

doi:10.3892/etm.2012.613

Construction of a novel vector expressing the fusion suicide gene yCDglyTK and hTERT-shRNA and its antitumor effects

Authors:
- Jia Li
- Guiying Zhang
- Ting Liu
- Huan Gu
- Lu Yan
- Bolin Chen
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Affiliations: Department of Gastroenterology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, P.R. China
Published online on: June 18, 2012 https://doi.org/10.3892/etm.2012.613
Pages: 442-448

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Abstract

This study aimed to construct a novel recombinant expression vector, pcDNA3.1(-)hTERT-shRNA/yCDglyTK. Its bioactivity and antitumor effects were investigated in the SGC7901 human gastric cancer cell line. Interfering RNA (RNAi) targeting human telomerase reverse transcriptase (hTERT) was applied to construct the pYr1.1-hTERT-shRNA vector. The shRNA expression cassette (including U6 promoter) was subcloned into the pcDNA3.1(-)CV-yCDglyTK vector to build a new vector, pcDNA3.1(-)hTERT-shRNA/yCDglyTK, which was identified by restriction enzyme digestion and gene sequencing. All the plasmids were delivered into SGC7901 cells using calcium phosphate nanoparticles (CPNPs). Expression of yCDglyTK and hTERT was detected by immunofluorescence, real-time PCR and western blot analysis. MTT assays were applied to measure the cytotoxic effect of the plasmids with 5-fluorocytosine (5-FC). Cell apoptosis was detected by flow cytometry. Restriction enzyme digestion and gene sequencing confirmed that the recombinant vector pcDNA3.1(-)hTERT-shRNA/yCDglyTK had been successfully constructed. Immunofluorescence, real-time PCR and western blot analysis showed that yCDglyTK was expressed, and that hTERT expression was inhibited in cells transfected with the recombinant vector. The cells transfected with the recombinant vector were the most sensitive to 5-FC and the apoptosis rates of the cells were also increased. The pcDNA3.1(-)hTERTshRNA/yCDglyTK vector was constructed successfully; it was confirmed that targeting hTERT through RNAi could synergize with suicide gene therapy.

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