Abstract:

BACKGROUND: The activation of telomerase is closely related to the occurrence and development of malignant tumor. While telomerase reverse transcriptase is the critical subunit of telomerase and now it is a hot point in the field of cancer research. RNA interference which is a kind of effective and specific gene blocking method has been widely used in cancer and virus research area.
OBJECTIVE: To construct the human telomerase reverse transcriptase-targeted small hairpin RNA-expressing plasmid (hTERT-targeted shRNA-expressing plasmid) system, and to observe its effect on hTERT expression and telomerase activity in breast cancer cells T47D.
METHODS: Sequence of hTERT-targeted shRNA was designed based on the mRNA sequence of hTERT which was obtained from the Genbank. They were recombined with the plasmid pBAsi-hU6-Neo (BamHI/HindⅢ) which is immune to antibiotic G418, and then those plasmids were identified by gene sequencing to make sure they were correctly connected. Then those plasmids were transfected into breast cancer cells T47D with liposome and those cells expressing shRNA were selected by G418.
RESULTS AND CONCLUSION: The hTERT-targeted shRNA-expressing plasmids were successfully constructed, which was proved by gene sequencing. Then the pBAsi-hU6-Neo recombined plasmids were transfected into T47D cells, and the successfully transfected cells were selected with G418. Reverse transcription-PCR and western blot showed that the expression of the trasfected hTERT was significantly decreased both on mRNA and protein levels (P < 0.01), and tartrate resistant acid phosphatase-enzyme-linked immunosorbent assay showed that the telomerase activity of the TERT in the experimental group was decreased significantly (P < 0.01). It showed that the hTERT-targeted shRNA-expressing plasmids were successfully constructed, and the designed small interferencing RNA can effectively block the expression of hTERT and then inhibit the telomerase activity.