AIM: To investigate the effects of antisense human telomerase RNA (hTR)on the biologic behavior of human gastric cancer cell line: MKN-45 by gene transfection and its potential role in the gene therapy of gastric canc...AIM: To investigate the effects of antisense human telomerase RNA (hTR)on the biologic behavior of human gastric cancer cell line: MKN-45 by gene transfection and its potential role in the gene therapy of gastric cancer. METHODS: The hTR cDNA fragment was cloned from MKN-45 through RT-PCR and subcloned into eukaryotic expression vector (pEF6/V5-His-TOPO) in cis-direction or trans-direction by DNA recombinant methods. The constructed sense, antisense and empty vectors were transfected into MKN-45 cell lines separately by lipofectin-mediated DNA transfection technology. After drug selection, the expression of antisense hTR gene in stable transfectants and normal MKN-45 cells was detected by RT-PCR, the telomerase activity by TRAP, the apoptotic features by PI and Hoechst 33258 staining, the cell cycle distribution by flow cytometry and the population doubling time by cell counting. Comparison among the stable transfectants and normal MKN-45 cells was made. RESULTS: The sense, antisense hTR eukaryotic expression vectors and empty vector were successfully constructed and proved to be the same as original design by restriction endonuclease analysis and sequencing. Then, they were successfully transfected into MKN-45 cell lines separately with lipofectin. The expression of antisense hTR gene was only detected in MKN-45 cells stably transfected with antisense hTR vector (named as MKN-45-ahTR) but not in the control cells. In MKN-45-ahTR, the telomerase activity was inhibited by 75%, the apoptotic rate was increased to 25.3%, the percentage of cells in the G0/G1 phase was increased to 65%, the proliferation index was decreased to 35% and the population doubling time was prolonged to 35.3 hours. However, the telomerase activity, the apoptotic rate, the distribution of cell cycle, the proliferation index and the population doubling time were not different among the control cells. CONCLUSION: Antisense hTR can significantly inhibit telomerase activity and proliferation of MKN-45 cells and induce cell apoptosis. Antisense gene therapy based on telomerase inhibition can be a potential therapeutic approach to the treatment of gastric cancer.展开更多
Background The 3q26 chromosome region, where the human telomerase RNA gene （hTERC） is located, is a biomarker for cervical cancer and precancerous lesions. The aim of this study was to confirm the value of measurin...Background The 3q26 chromosome region, where the human telomerase RNA gene （hTERC） is located, is a biomarker for cervical cancer and precancerous lesions. The aim of this study was to confirm the value of measuring hTERC gene gain in predicting the progression of cervical intraepithelial neoplasia grade Ⅰ or Ⅱ （CIN-Ⅰ and -Ⅱ, respectively） to CIN-Ⅲ and cervical cancer. Methods Liquid-based cytological samples from 54 patients with CIN-Ⅰ or CIN-Ⅱ lesions were enrolled in this study. Follow-up was performed with colposcopy and biopsy within 24 months after the diagnosis of CIN-Ⅰ or CIN-Ⅱ. Copy numbers of the hTERC gene were measured by fluorescence in situ hybridization with a dual-color probe mix containing the hTERC gene probe （labeled red） and the control, the chromosome 3 centromere-specific probe （labeled green）.Results All patients whose lesions progressed from CIN-Ⅰ or CIN-Ⅱ to CIN-Ⅲ displayed a gain of the hTERC gene, whereas patients where the hTERC gene was not amplified did not subsequently progress to CIN-Ⅲ or cervical cancer. The signal ratio pattern per cell was recorded as N：N （green： red）. The numbers of cells with the signal ratio pattern of 4：4 or N：≥5 in patients whose lesions progressed to CIN-Ⅲ were significantly higher than those whose lesions did not progress. Significantly, none of the patients with a 4：4 signal ratio pattern regressed spontaneously.Conclusions In conclusion, measurement of hTERC gene gain in CIN-Ⅰ or CIN-Ⅱ patients using liquid-based cytological samples could be a useful biomarker to predict the progression of such cervical lesions. In addition, a 4：4 or N：≥5 signal ratio pattern may indicate the unlikeness of spontaneous regression of CIN-Ⅰ or CIN-Ⅱ lesions.展开更多
基金the National Natural Science Foundation of China,No.39770725
文摘AIM: To investigate the effects of antisense human telomerase RNA (hTR)on the biologic behavior of human gastric cancer cell line: MKN-45 by gene transfection and its potential role in the gene therapy of gastric cancer. METHODS: The hTR cDNA fragment was cloned from MKN-45 through RT-PCR and subcloned into eukaryotic expression vector (pEF6/V5-His-TOPO) in cis-direction or trans-direction by DNA recombinant methods. The constructed sense, antisense and empty vectors were transfected into MKN-45 cell lines separately by lipofectin-mediated DNA transfection technology. After drug selection, the expression of antisense hTR gene in stable transfectants and normal MKN-45 cells was detected by RT-PCR, the telomerase activity by TRAP, the apoptotic features by PI and Hoechst 33258 staining, the cell cycle distribution by flow cytometry and the population doubling time by cell counting. Comparison among the stable transfectants and normal MKN-45 cells was made. RESULTS: The sense, antisense hTR eukaryotic expression vectors and empty vector were successfully constructed and proved to be the same as original design by restriction endonuclease analysis and sequencing. Then, they were successfully transfected into MKN-45 cell lines separately with lipofectin. The expression of antisense hTR gene was only detected in MKN-45 cells stably transfected with antisense hTR vector (named as MKN-45-ahTR) but not in the control cells. In MKN-45-ahTR, the telomerase activity was inhibited by 75%, the apoptotic rate was increased to 25.3%, the percentage of cells in the G0/G1 phase was increased to 65%, the proliferation index was decreased to 35% and the population doubling time was prolonged to 35.3 hours. However, the telomerase activity, the apoptotic rate, the distribution of cell cycle, the proliferation index and the population doubling time were not different among the control cells. CONCLUSION: Antisense hTR can significantly inhibit telomerase activity and proliferation of MKN-45 cells and induce cell apoptosis. Antisense gene therapy based on telomerase inhibition can be a potential therapeutic approach to the treatment of gastric cancer.
文摘Background The 3q26 chromosome region, where the human telomerase RNA gene （hTERC） is located, is a biomarker for cervical cancer and precancerous lesions. The aim of this study was to confirm the value of measuring hTERC gene gain in predicting the progression of cervical intraepithelial neoplasia grade Ⅰ or Ⅱ （CIN-Ⅰ and -Ⅱ, respectively） to CIN-Ⅲ and cervical cancer. Methods Liquid-based cytological samples from 54 patients with CIN-Ⅰ or CIN-Ⅱ lesions were enrolled in this study. Follow-up was performed with colposcopy and biopsy within 24 months after the diagnosis of CIN-Ⅰ or CIN-Ⅱ. Copy numbers of the hTERC gene were measured by fluorescence in situ hybridization with a dual-color probe mix containing the hTERC gene probe （labeled red） and the control, the chromosome 3 centromere-specific probe （labeled green）.Results All patients whose lesions progressed from CIN-Ⅰ or CIN-Ⅱ to CIN-Ⅲ displayed a gain of the hTERC gene, whereas patients where the hTERC gene was not amplified did not subsequently progress to CIN-Ⅲ or cervical cancer. The signal ratio pattern per cell was recorded as N：N （green： red）. The numbers of cells with the signal ratio pattern of 4：4 or N：≥5 in patients whose lesions progressed to CIN-Ⅲ were significantly higher than those whose lesions did not progress. Significantly, none of the patients with a 4：4 signal ratio pattern regressed spontaneously.Conclusions In conclusion, measurement of hTERC gene gain in CIN-Ⅰ or CIN-Ⅱ patients using liquid-based cytological samples could be a useful biomarker to predict the progression of such cervical lesions. In addition, a 4：4 or N：≥5 signal ratio pattern may indicate the unlikeness of spontaneous regression of CIN-Ⅰ or CIN-Ⅱ lesions.
