Construction of antisense hTERT retroviral vector and its inhibition on the proliferation of HT29 colorectal cancer cells

Objective: To construct the antisense human telomerase reverse transcriptase (hTERT) cDNA retroviral vector, investigate the inhibition of telomerase activity and celluler proliferation in colorectal cancer cells HT29 by it treatment. and explore the possibility of the hTERT as a target for colorectal cancer gene therapy. Methods:We used RT PCR amplify the hTERT mRNA 5′ region 835bp fragment, and cloned the hTERT fragment into pLXSN retroviral vector in sense and antisense orientations. The replication deficient retroviruses were by obtained transfecting the packing cells of PT67, and then infecting the HT29 colorectal cancer cells. The hTERT protein expression was detected by Western blot, telomerase activity was determined by telomerase repeat amplification protocol (TRAP), cell proliferation was investigated by invert microscope and growth curve, and the apoptosis was characterized by flow cytometry and DNA electrophoresis. Results: Compared with the sense hTERT transduced HT29, the hTERT protein expression, telomerase activity and cell proliferation of HT29 were significantly inhibited and apoptosis occurred after antisense hTERT transduction. Conclusion: The transduction of antisense hTERT can significantly inhibit the hTERT expression, suppress telomerase activity, arrest the cell growth and accelerate apoptosis in HT29. hTERT is a potential target for colorectal cancer gene therapy.