Construction and identification of human telomerase reverse transcriptase-targeted small hairpin RNA-expressing plasmid and its stable transfected cervical cancer line Caski

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 cervical cancer cells Caski. Methods: Sequence of hTERT-targeted shRNA was designed by Designer3. 0( Genepharma) based on the mRNA sequence of hTERT which was obtained from the Genbank. They were recombined with the plasmid pGLV3 / H1 / GFP + Puro,and then those plasmids were identified by gene sequencing to make sure they were correctly connected. Then those plasmids were transfected into cervical cancer cells Caski and fluorescence expression was observed. The expression of hTERT mRNA and protein was identified by RT-PCR and Western blot respectively. The telomerase activity was detected by TRAP-ELISA. Results: The hTERT-targeted shRNA-expressing plasmids were successfully constructed,which was proved by gene sequencing. Then the pGLV3 / H1 / GFP + Puro recombined plasmids modified by fluorescein was synthesized and transfected into Caski cells,the transfection efficiency of shRNA was evaluated by calculating the ratio of fluorescent cells to total cells. Observed by fluorescence microscopy,24 h after transfection,Caski cells began to emit green fluorescence,72 h after transfection,a large number of fluorescent expression,while there was no expression in Caski cells that stably transfected with empty vector. Caski cells after transfection were detected and the results showed that there were three chains could significantly inhibit the expression of hTERT mRNA and protein levels and significantly reduced telomerase activity,72H-TERT-1515 was the strongest,inhibition rate of hTERTmRNA and protein was 75. 0% and 70. 3%,respectively,telomerase activity decreased 74. 6%. Conclusion: The hTERT-targeted shRNA-expressing plasmids have been successfully constructed,and the designed small interferencing RNA can effectively block the expression of hTERT and then inhibit the telomerase activity. It provides a basis for further study that the role of telomerase in pathogenesis and interference in vitro of gene therapy treatment of cervical cancer