Molecular cloning of hTERT promoter and analysis of specific transcriptional activities in human cancer cells
YANG Shaomin, ZHANG Bo, LIAO Songlin, et al. (Department of Pathology, Health Science Center of Peking University, Beijing 100083)
Objective To probe the activation mechanism of hTERT gene in cancer formation based on cloning its upstream regulating region and observing the specific transcription activity in verious transiently transfected human cancer cells and human diploid cells. Methods The transcription regulation region of hTERT in HeLa and PG cells were amplified by polymerase chain reaction and confirmed by DNA sequencing, then were reconstructed into luciferase reporter vectors and transiently transfected into HeLa, PG, 293 and normal human diploid 2BS cell. The transcription activities were determined by measuring the luciferase activities. Results The 636bp (-531～+90) and 950bp(-531～+404) hTERT transcription regulation regions in HeLa and PG cells have the same sequences with the hTERT promoter registered in Genbank. These transcription regulation regions were cloned into luciferase reporter plasmid to generate the recombinants pGL2 636 and pGL2 950. Meanwhile, pGL2 X (-531～-272) and pGL2 S (the first intron region) were reconstructed. After transient transfection, results showed that luciferase activity increased dramatically in cancer cells transfected with pGL2 636 and pGL2 950, whereas no luciferase activity was identified in human diploid 2BS cells. The transcription activities of pGL2 S and pGL2 X decreased significantly in telomerase positive tumor cells, and that of pGL2 S was slightly higher than pGL2 X. Conclusion In human HeLa and PG cells, the transcription regulation regions of hTERT gene have no gene mutation and are highly conservative. The transcriptional activation of hTERT correlated with the expression of telomerase and cancer transformation. The -272bp upstream region of hTERT gene was the core regulatory region, and the first intron had only weak transcriptional activity. The transcription regulation region of hTERT had varying activities in different cancer cells.
【CateGory Index】： R730.2