Construction of an expression vector carrying short hairpin RNA targeting hTERT gene and its effects on breast cancer cell telomerase activity and proliferation in vivo / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To construct a RNA interference expression vector targeting human telomerase reverse transcriptase gene (hTERT) gene and investigate its effects on telomerase activity and proliferation in breast cancer cells in vitro.</p><p><b>METHODS</b>The shRNA sequences targeting hTERT gene were designed and recombined into pSuper-retro-puro vector. The breast cancer cell lines MCF-7 and MDA-MB231 were transfected with the recombined vector, and the telomerase activity of the cells was tested by telomerase repeat sequence amplification-enzyme linked immunosorbent assay (TRAP-ELISA). The proliferation of the transfected cells was assessed using MTT and soft-agar clone formation assays.</p><p><b>RESULTS</b>The recombinant plasmids pSuper-retro-puro-TERT RNAi#1 and #2 were successfully constructed as confirmed by enzymatic digestion and DNA sequencing. The telomerase activity in the transfected breast cancer cells were down-regulated significantly as compared with that in negative control cells (Plt;0.005). The transfection resulted in significant inhibition of the proliferation of both MCF-7 and MDA-MB231 cells as detected by MTT assay (Plt;0.05) and soft agar clone formation assay (Plt;0.001).</p><p><b>CONCLUSION</b>Transfection with the recombinant plasmid containing the shRNA targeting hTERT gene can down-regulate telomerase activity and inhibit proliferation of breast cancer cells in vitro, suggesting the potential of gene therapy targeting telomerase in the treatment of breast cancer.</p>