脱氧核酶对端粒酶RNA的切割和乳腺癌细胞凋亡相关基因表达的作用  

Cleavage of telomerase RNA component by two DNAzymes and their effects on expression of two apoptosis-related genes in human mammary cancer cells

作　　者：赵家明[1] 李明意[2] 杨展[1] 李震[1] 张毅[1] 

机构地区：[1]广东医学院附属医院中心实验室,广东湛江524001 [2]广东医学院附属医院普通外科,广东湛江524001

出　　处：《第一军医大学学报》2005年第6期638-642,共5页Journal of First Military Medical University

基　　金：广东省卫生厅青年基金(B1997119)~~

摘　　要：目的探讨脱氧核酶对端粒酶RNA组分(hTR)的切割作用及对人乳腺癌细胞凋亡相关基因bcl-2和bax表达的影响.方法针对hTR基因的核苷酸序列,合成'10～23'型脱氧核酶及其类似物,提取总RNA在体外切割hTR RNA;转染乳腺细胞后,用RT-PCR扩增hTR基因片段,用PCR-ELISA法测定端粒酶活性,用RT-PCR和荧光免疫测定凋亡相关基因bcl-2和bax的表达.结果未经修饰的脱氧核酶DzT和在DzT的3′末段添加倒位连接T碱基的脱氧核酶DzTi在体外能够有效地切割hTRRNA;在转染乳腺癌细胞后,DzTi比DzT对hTRRNA表现出更强的切割作用,DzTi能够显著地降低乳腺癌细胞端粒酶活性(P＜0.05),但对乳腺癌细胞的生长和细胞凋亡相关基因bcl-2和bax基因没有明显的影响(P＞0.05).DzT和DzTi的催化中心的一个碱基被替换后形成的脱氧寡核苷酸DzT′和DzTi′在胞外和胞内均不表现对hTR RNA的切割作用.结论人工合成的脱氧核酶确实能够高效特异地切割端粒酶hTR RNA,并降低乳腺癌细胞的端粒酶活性.作为一种新发现的催化性核酸,脱氧核酶在肿瘤的基因治疗领域中可能具有极其重要的应用价值.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.

关 键 词：催化性DNA 端粒酶 乳腺肿瘤 bcl-2 BOX 

分 类 号：R737.9[医药卫生—肿瘤] Q523[医药卫生—临床医学]