脱氧核酶对端粒酶hTERT mRNA的切割以及对鼻咽癌细胞凋亡相关基因的影响  

Synthesized DNAzymes cleaves the mRNA of human telomerase reverse transcriptase (hTERT) and impacts on expressions of apoptosis related genes in nasopharyngeal carcinoma cells

作　　者：赵家明[1] 李明意[2] 李震[1] 杨展[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肝胆外科,广东湛江524001

出　　处：《广东医学院学报》2005年第3期242-245,共4页Journal of Guangdong Medical College

摘　　要：目的:探讨脱氧核酶对端粒酶蛋白亚基hTERTmRNA的切割作用及对人鼻咽癌细胞凋亡相关基因表达的影响。方法:针对hTERT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hTERTmRNA;转染鼻咽癌细胞后,用RT-PCR扩增hTERT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hTERTmRNA;在转染鼻咽癌细胞后,DzTi比DzT对hTERTmRNA表现出更强的切割作用,DzTi能够显著地降低鼻咽癌细胞端粒酶活性(P<0.01)、抑制鼻咽癌细胞的生长(P<0.05)和bcl-2基因的表达(P<0.05),上调bax基因的表达(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'在体外和胞内均不表现对hTERTmRNA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hTERTmRNA,并降低鼻咽癌细胞的端粒酶活性,促进鼻咽癌细胞凋亡。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将具有极其重要的应用价值。Objective:To test the function of synthesized DNAzymes to cleave the mRNA of human telomerase reverse transcriptase (hTERT) and to impact on expressions of apoptosis related genes in a human nasopharyngeal carcinoma cell line (CNE-2Z). Methods:Two 10～23 DNAzymes (DzT and DzTi, targeting against hTERT mRNA and their analogues (DzT' and DzTi') were synthesized; the cleavage ability of DNAzymes was tested in vitro with mRNA from CNE-2Z cells; the in vivo hTERT fragment and telomerase activity from the CNE-2Z cells transfected with the DNAzymes were tested by RT-PCR and PCR-ELISA assay; the biological effect of the DNAzymes on the expression of two apoptosis related genes (bcl-2 and bax) was evaluated with RT-PCR and immuno-fluorescent assay. Results:Both DzT (the unmodified DNAzyme) and DzTi (the modified form that was added with an inverted thymidine at 3'-end) could effectively cleave hTERT mRNA in vitro; in transfected CNE-2Z cells, DzTi exhibited a higher efficiency than did DzT in cleavage ability, to down-regulate the telomerase activity (P<0.01) and the expression of bcl-2 gene (P<0.05), inhibit the cell growth (P<0.05), and up-regulate the expression of bax gene (P<0.05); DzT' and DzTi' (analogues with a base displacement in the conserved catalytic motif from DzT and DzTi, respectively) did not exhibit any notable cleavage effect on hTERT mRNA either in vitro or in cells. Conclusions:The synthesized DNAzymes could effectively and specifically cleave hTERT mRNA, decrease the telomerase activity and induce apoptosis in CNE-2Z cells, and might have anti-tumor potential.

关 键 词：脱氧核酶 端粒酶 鼻咽肿瘤 细胞凋亡 

分 类 号：R739.6[医药卫生—肿瘤] Q523[医药卫生—临床医学]