构建MicroRNA-29a和microRNA-29c腺病毒并探讨其对膀胱癌细胞增殖能力的影响  

MiR-29a and miR-29c Recombinant Adenovirus Vector Regulate Proliferation of Human Bladder Cancer Cells

作　　者：范砚茹[1] 杜红飞[1] 宋学东[1] 罗春丽[1] 

机构地区：[1]重庆医科大学检验医学院,重庆400016

出　　处：《中国细胞生物学学报》2014年第2期184-189,共6页Chinese Journal of Cell Biology

摘　　要：构建miRNA-29a/c的重组腺病毒并观察其对膀胱癌T24细胞增殖能力的调控。以人全基因组DNA为模板,PCR扩增miR-29a、miR-29c,克隆至腺病毒穿梭载体pAdtrace-TO4-CMV。重组穿梭载体经pme I线性化后与腺病毒骨架质粒pAdEasy-1共转化感受态大肠杆菌BJ5183,通过同源重组获得重组腺病毒质粒pAdEasy-1-miR-29a、pAdEasy-1-miR-29c,pac I线性化后转染HEK-293细胞,进行包装和扩增。实时荧光定量PCR检测感染腺病毒的膀胱癌T24细胞中miR-29a、miR-29c的表达水平,并利用CCK-8实验检测细胞增殖能力。经DNA测序和限制性内切酶分析显示,重组腺病毒质粒pAdEasy-1-miR-29a、pAdEasy-1-miR-29c构建成功;感染腺病毒Ad-miR-29a和Ad-miR-29c后,经实时荧光定量PCR检测,膀胱癌细胞中miR-29a、miR-29c表达显著增高(P<0.01);过表达miR-29a/c后的CCK-8实验显示,细胞增殖能力明显低于对照组(P<0.05)。以上说明已成功构建miR-29a、miR-29c腺病毒,过表达miR-29a/c可抑制膀胱癌细胞的增殖。To construct recombinant adenovirus vector containing human miR-29a and miR-29c and to determine its effect on the proliferation in human bladder cancer T24 cells, the PCR product containing miR-29a/c was amplified from human genomic DNA and inserted into the adenoviral shuttle vector pAdTrace-TO4-CMV. Then, the recombinant shuttle plasmid linearized by prne I was co-transformed into competent E. coli. B J5183 with the adenovi- ral backbone plasmid pAdEasy-1. Then, the recombinant adenoviral DNA was transfected into HEK293 cells, packed and amplified miR-29a and miR-29c adenoviruses. T24 cells were infected by Ad-miR-29a and Ad-miR-29c. The expression of mature miR-29aJc was detected by Real-time PCR. The cell proliferation was detected by CCK-8 assay before and after infected miR-29a/c. Real-time PCR showed that miR-29a and miR-29c significantly up-regulated in T24 cells infected with Ad-miR-29a and Ad-miR-29c （P〈0.01）. CCK-8 assay showed that cell proliferation was significantly depressed after infection （P〈0.05）. The adenovirus vector Ad-miR-29a and Ad-miR-29c were constructed successfully and the over-expression ofmiR-29c inhibited the proliferation ofT24 cells.

关 键 词：MICRORNA 29a MICRORNA 29c 腺病毒 膀胱癌 细胞增殖 

分 类 号：R737.14[医药卫生—肿瘤]