Cloning of human telomerase reverse transcriptase gene promoter and analysis of specific activities in human hepatocellular carcinoma cells

Objective:To clone the hTERT gene promoter and detect its transcriptional activities in various hepatocellular carcinoma cell lines and in human fibroblast cells in order to make groundwork for the targeting gene therapy for human hepatocellular carcinoma.Methods:The fragment of hTERT promoters were acquired by PCR amplification and were cloned into the reporter plasmid pGL3-Basic which contains a luciferase gene.The constructed eukaryotic expression plasmid pGL3-hTERT in which the expression of the luciferase were drived by hTERT promoter were transfected into three HCC cell lines and the primary fibroblast cells.At 24 hours post transfection (p.t.),the activity of the luciferase was determined with Dual-Luciferase Reporter Assay System.A pGL3-AFP containing luciferase gene controlled by the AFP promoter was used as positive control and a pGL3-Basic vector which have no promoter in front of the luciferase gene was used as negative control.Results:The hTERT promoter was successfully cloned into the eukaryotic reporter vector,pGL3-Basic and gained the pGL3-hTERT vector.The hTERT promoters showed high transcriptional activities in all the three HCC cell lines but no transcriptional activities in the primary fibroblast cells.The transcriptional activity of the hTERT promoter was much higher than AFP promoter(14～30 times).Conclusion:Our data revealed that the hTERT promoter possesses the tumor-spesific high transcrioptional activity in all the three established HCC cell lines.It may serve as a useful tool for transcriptional targeting of hepatocellular carcinoma gene therapy.