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HSV gene regulation of hTERT promoter

Chinese papers League finishing. 【Abstract】 AIM: To observe the killing effect of HSVtk / GCV system on cervical carcinoma cells under the control of hTERT ( human telomerase reverse transcriptase) core promoter. METHODS: An eukaryotic expression vector containing HSVtk gene under the control of hTERT core promoter was constructed and was transfected into cervical carcinoma cells (HeLa) and vessel endothelial cells (ECV304) by liposome method. Transfected cells were then selected with G418 and RTPCR was used to detect the tk gene expression in HeLa cells and ECV304 cells. After GCV was added, flow cytometry method were applied to investigate its cell killing effect. RESULTS: pCIneo / hTERTtk / GCV system under the control of hTERT induced the apoptosis in more than 36.7% of cervical carcinoma cells, but not in normal vessel endothelial cells. CONCLUSION: hTERT gene core promoter is tumorspecific and may be useful in tk gene therapy of cervical carcinoma and in reducing the side effects of the therapy . 【Keywords】 telomerase; promoter regions (genetics); cervix neoplasms; tk gene Abstract Objective: To study the human telomerase reverse transcriptase (hTERT) gene core promoter Sub-regulation of human herpes simplex virus thymidine kinase gene / ganciclovir (HSVtk / GCV) system on cervical cancer cells in vitro. METHODS: regulation of hTERT gene promoter tk gene eukaryotic expression vector pCIneo / hTERTtk, were transfected by liposome cervical carcinoma cells (HeLa) and normal endothelial cells (ECV304), neomycin (G418) screened positive for clonal expansion. RTPCR method by comparing the two tk gene expression in cells; given before drug ganciclovir (GCV), with flow cytometry hTERT regulation of HSVtk / GCV system on two types of cells in vitro. Results: hTERT promoter under the control of the HSVtk / GCV system for cervical cancer HeLa cells significantly killing effect, so that 36.7% of the cells; and ECV304 effect on normal cells is not obvious. Conclusion: hTERT promoter control of the tk gene therapy is a kind of cancer specific treatment, is expected to address the specific tumor gene therapy of killing and reduced side effects and other issues. Key words telomerase enzyme; promoter (genetics); cervical cancer; tk gene 0 Introduction higher mortality rates of cervical cancer is a malignant tumor, conventional treatment with surgery, chemotherapy, radiotherapy based, but not late surgery and radiotherapy and chemotherapy were not sensitive to 5 a survival was still not ideal. with molecular biology and related technology is developing rapidly, suicide gene therapy in cancer therapy achieved a certain effect, the use of human herpes simplex virus thymidine kinase gene / ganciclovir (human simplex virusthymidine kinase / ganciclovir, HSVtk / GCV) to ganciclovir (ganciclovir,[link widoczny dla zalogowanych], GCV) and other cytotoxic prodrug into a product of phosphoric acid, which prevents cell DNA synthesis, killing tumor cells [1-2]. have found that the lack of specificity, tk gene-modified cells in normal tissues after treatment by the former drug along with the tumor cells are killed, resulting in the destruction of normal tissue. hTERT gene in 90% of the human tumor tissue was over-expression [3], with a significant tumor-specific resistance. We used cloned hTERT gene promoter, using its unique in tumor cells to control the promoter activity of tumor-specific expression of HSVtk way to achieve its specific killing of tumor cells in the role of. 1 Materials and methods 1.1 Materials Taq DNA polymerase, restriction enzymes and gel electrophoresis and used in marker DL2000 were purchased from TaKaRa Company DGL2000; T4 DNA ligase purchased from Promega Corporation of Japan; plasmid extraction and purification kit was purchased from Beijing days for Time Inc.; LipofectamineTM2000 transfection kit purchased from Introvigen Company; containing human telomerase reverse transcriptase (hTERT) gene and the tk gene of pGL3 Basic plasmid by the Fourth Military Medical University, Department of Otolaryngology, Han Ming Tang Kun gift; pCIneo plasmid by the Fourth Military Medical University Hospital, Department of Nephrology Yang Jie Tang gifts; GCV were purchased from Guangdong Livzon; E. coli strains JM109,[link widoczny dla zalogowanych], HeLa cervical cancer cells and umbilical vein ECV304 endothelial cells by the Fourth Military Medical University, Tang Du Hospital, providing clinical trial subjects; flow cytometry,[link widoczny dla zalogowanych], Coulter, USA. 1.2 Methods 1.2.1hTERT gene primers were designed and synthetic design hTERT gene promoter primer restriction sites for the Bgl Ⅱ and Hind Ⅲ, from the Shanghai GeneCore biotechnology companies synthesized. upstream primer: 5'ggg aga tct agt gga ttc gcg ggc aga ga 3 '; downstream primer: 5' ggg aag ctt agg gct tcc cac gtg cgc ag3 '. to the plasmid containing the hTERT promoter as a template for PCR amplification,[link widoczny dla zalogowanych], cycle parameters: 94 ℃ 2 min; 94 ℃ 30 s, 67 ℃ 30 s, 72 ℃ 40 s, 30 cycles; 72 ℃ 1 min. PCR products were analyzed by 15 g / L agarose gel electrophoresis, recovered by gel extraction kit hTERT promoter fragments. 1.2.2 plasmid will be the hTERT by Bgl Ⅱ fragment and Hind Ⅲ restriction enzyme digestion, by both ends of the Bgl Ⅱ and Hind Ⅲ site of hTERT promoter fragments, to connect it into the pGL3tk plasmid between the corresponding restriction sites to obtain plasmid containing hTERTtk pGL3. plasmid of the above with Bgl Ⅱ and Xba Ⅰ restriction enzyme digestion, by hTERTtk gene fragments into pCIneo plasmid with its corresponding restriction enzyme sites between the hTERT promoter regulated by tk gene eukaryotic expression vector pCIneo / hTERTtk, and the digestion method identification. 1.2.3 plasmid transfected HeLa cells and ECV304 cell recovery, cultured in 100 mL / L calf serum basic medium necessary for Darfur Bo Keshi (DMEM) in the. transfection ago 1 d, cells were trypsinized and counted, the cell containing 2 mL of serum in the ceiling, without antibiotics, the normal growth of the culture medium (3 × 105 cells per well), so that it can be transfected at 80% coverage . inoculated with 2 holes for each cell were transfected with pCIneo / hTERTtk and pCIneo two plasmids were named HeLa / hTERTtk, HeLa / 0, ECV304/hTERTtk and ECV304 / 0. For each hole cell, lipid per 10 uL body added 4 μg DNA, in serum-free DMEM medium mixing, 37 ℃, 50 mL / L CO2 and incubated for 5 h, replace medium with serum were incubated for 24 h, instead of culture medium containing G418, G418 concentration to 500 μg / mL, continue to screen monoclonal picked after 12 d, and expand training. During the medium was changed every 3 d 1, by stable transfection of two plasmids in the cell. 1.2.4RTPCR were Extraction of ECV304 transfected HeLa cell genome and the total RNA, conventional method of reverse transcription cDNA, and used as templates for the core tk gene sequence PCR. upstream primer: 5'gca tgg ctt cgt acc cct gcc atc3 '; downstream primer : 5'gcg tta gcc tcc ccc atc tcc cgg3 '. cycle parameters: 94 ℃ 3 min; 94 ℃ 40 s, 60 ℃ 40 s, 72 ℃ 70 s, 30 cycles; 72 ℃ 8 min. PCR products were analyzed by 15 g / L agarose gel electrophoresis of 0.5 h, photographed under UV light. 1.2.5 flow cytometry different cells transfected with vector named: HeLa / hTERTtk, HeLa / 0, ECV304/hTERTtk and ECV304 / 0, 2 × 105, respectively, according to the number of seeded in 25 cm2 culture flasks, 37 ℃, 50 mL / L CO2 incubator for 24 h, incubated with GCV (1 mg / mL) culture medium, incubated for collected after 72 h 1 × 106 cells, first washed 2 times with PBS, and then ethanol fixed and sent for analysis by flow cytometry, each cell to send 5 copies. centrifugation prior to testing to ethanol, PI staining Anhui 15 min. mercury laser excitation wavelength of 488 nm, the results of using Elite 4.0 software, and DNA Multictycle analysis. Statistical analysis: x ± s data,[link widoczny dla zalogowanych], said statistical analysis with SPSS11.0 software, a number of samples of the comparative analysis of variance; twenty-two comparison between the mean test with SNKq. P <0.01 was considered statistically significant.