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

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pmeI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，pacI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伊〈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。

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

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.