脱氧核酶对cip1/waf1 mRNA的切割及对人绒毛膜癌细胞凋亡相关基因表达的影响  

Cut of human cip1/waf1 mRNA by two synthesized DNAzymes and their effects on two apoptosis-related genes in a human choriocarcinoma cell line

作　　者：赵家明[1] 杨展[1] 李震[1] 李明意[1] 吴柱国[1] 张毅[1] 何惠娟[1] 

机构地区：[1]广东医学院附属医院中心实验室,湛江524001

出　　处：《第三军医大学学报》2005年第7期636-639,共4页Journal of Third Military Medical University

基　　金：广东省卫生厅青年基金资助项目 (B1997119)~~

摘　　要：目的　探讨脱氧核酶对cip1/waf1mRNA的切割作用及对人绒毛膜癌细胞凋亡相关基因表达的影响。方法　针对cip1/waf1基因的核苷酸序列,合成“10～2 3”型脱氧核酶及其类似物,提取总RNA在体外切割cip1/waf1mRNA ;转染绒毛膜癌细胞后,用RT PCR扩增和荧光免疫方法测定cip1/waf1和凋亡相关基因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cip1/waf1mRNA ;在转染绒毛膜癌细胞后,DzTi比DzT对cip1/waf1mRNA表现出更强的切割作用,能显著地降低细胞内cip1/waf1蛋白(p2 1cip1/waf1蛋白或p2 1蛋白)水平(P <0 0 1) ,下调bax基因的表达(P <0 0 5 ) ,但对bcl 2基因的表达和细胞的生长未表现出显著的影响(P >0 0 5 )。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cip1/waf1mRNA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cip1/waf1mRNA ,并能上调凋亡促进基因bax的表达,表明脱氧核酶确实能够通过切割cip1/waf1mRNA来参与细胞的凋亡过程。Objective To explore the cut of human cip1/waf1 mRNA by two DNAzymes and their effects on expression of two apoptosis-related genes in the human choriocarcinoma cell line Jar. Methods Two “10-23” DNAzymes (DzT and DzTi) targeted against cip1/waf1 mRNA and their analogues (DzT′ and DzTi′) were synthesized and used to cut cip1/waf1 mRNA in vitro and in Jar cells. The RT-PCR amplification of a cip1/waf1 DNA fragment and telomerase activity assay by PCR-ELISA were performed to assess the cutting efficiency. The biological effects of the DNAzymes were evaluated by measuring the expression of two apoptosis-related genes (bcl-2 and bax). Results The non-modified DNAzyme, DzT, and its modified form, DzTi, that had an added 3′-inverted thymidine, could effectively cut cip1/waf1 mRNA in vitro. After transfected into Jar cells, DzTi exhibited more powerful cutting ability than DzT and down-regulated the level of cip1/waf1 protein (p21cip1/waf1 or p21 protein) (P<0.01) and the expression of pro-apoptotic gene bax (P<0.05). Neither DzTi nor DzT displayed significant influence on the growth of the cells and the expression of antiapoptotic genes bcl-2. DzT′ and DzTi′, which derived respectively from DzT and DzTi with a base displacement in the conserved catalytic motif, did not exhibit notable cutting effects on cip1/waf1 mRNA either in vitro or in vivo. Conclusion The synthesized DNAzymes could cut cip1/waf1 mRNA with effectiveness and specificity and probably be involved in apoptosis via regulating apoptosis-related genes. As a newly found catalytic nucleic acid, DNAzymes will play an important role in gene regulation field.

关 键 词：催化性DNA cipl/waf1 绒毛膜癌 BCL-2 BAX 

分 类 号：Q556.4[生物学—生物化学] R394.2[医药卫生—医学遗传学]