[Cleavage of telomerase RNA component by two DNAzymes and their effects on expression of two apoptosis-related genes in human mammary cancer cells].
OBJECTIVE
To investigate the cleavage of telomerase RNA component (hTR) by two DNAzymes and their effects on the expression of two apoptosis-related genes in a human mammary cancer cell line (MCF-7).


METHODS
Two "10-23" DNAzymes (DzT and DzTi) targeted against hTR RNA and their analogues (DzT' and DzTi') were synthesized and used to cleave hTR RNA in vitro and in MCF-7 cells. Reverse transcriptional (RT) PCR amplification of a hTR DNA fragment and telomerase activity assay by PCR-enzyme-linked immunosorbent assay (ELISA) were performed to assess the cleavage efficiency. The biological effect of the DNAzymes was evaluated by measuring the expression of two apoptosis-related genes (bax-2 and bax).


RESULTS
The unmodified DNAzyme, DzT, and its modified form, DzTi, which had an 3'-inverted thymidine added, could effectively cleave hTR mRNA in vitro. After transfection into MCF-7 cells, DzTi exhibited more powerful cleavage ability than DzT, and down-regulated the activity of telomerase (P<0.05). However, neither DzTi nor DzT displayed significant effect on the growth of the cells (P>0.05) and the expression of bax-2 and bax (P>0.05). DzT' and DzTi', derived from DzT and DzTi respectively with a base displacement in the conserved catalytic motif, did not exhibit notable cleavage effect on hTR RNA either in vitro or in the cells.


CONCLUSIONS
The synthesized DNAzymes can effectively and specifically cleave hTR RNA and decrease the telomerase activity. As a newly found catalytic nucleic acid, DNAzymes will play an important role in gene therapy studies of tumors.
Zhao
Jia-ming
JM
Central Laboratory, Affiliated Hospital of Guangdong Medical College, Zhanjiang 524001, China. zyunyang@163.net
Li
Ming-yi
MY
Yang
Zhan
Z
Li
Zhen
Z
Zhang
Yi
Y
D017209:Apoptosis / Q000502:physiology*; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D021881:DNA, Catalytic / Q000494:pharmacology*; D005260:Female; D006801:Humans; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics; D016372:RNA, Antisense / Q000494:pharmacology*; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein / Q000096:biosynthesis / Q000235:genetics
D021881:DNA, Catalytic; D019253:Proto-Oncogene Proteins c-bcl-2; D016372:RNA, Antisense; D051028:bcl-2-Associated X Protein; D019098:Telomerase