[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].
OBJECTIVE
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.


METHODS
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.


RESULTS
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).


CONCLUSIONS
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.
Liu
Xiang-Xia
XX
Department of Plastic and Reconstructive Surgery, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China. liuxsha@mail.sysu.edu.cn
Yao
Chen
C
Zhang
Hui
H
Wang
San-Ming
SM
Wang
Shen-Ming
SM
D001943:Breast Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D005260:Female; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D019098:Telomerase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D012333:RNA, Messenger; D034741:RNA, Small Interfering; C509186:TERT protein, human; D019098:Telomerase