慢病毒介导的p21/Waf1基因沉默增强膀胱癌特异性溶瘤腺病毒的复制及对EJ细胞增殖的抑制作用  

Lentivirus-mediated p21/Waf1 silencing enhances replicative potential of bladder cancer-specific oncolytic adenovirus and its inhibition effect on proliferation of EJ cells

作　　者：石洁[1,2,3] 王志平 李晓娟[1] 杨兰[1] 秦晓东 王莉[5] 付生军 陶燕 

机构地区：[1]兰州大学第二医院输血科,甘肃兰州730030 [2]甘肃省泌尿系疾病重点实验室,甘肃兰州730030 [3]兰州大学生命科学院,甘肃兰州730000 [4]兰州兽医研究所,甘肃兰州730046 [5]兰州大学基础医学院药理教研室,甘肃兰州730000

出　　处：《肿瘤》2015年第10期1083-1091,共9页Tumor

摘　　要：目的 :沉默人膀胱癌EJ细胞中p21/Waf1基因的表达,并探讨p21/Waf1表达下调对膀胱癌特异性溶瘤腺病毒复制及对EJ细胞增殖的影响。方法 :设计并合成特异性针对p 21/Waf 1基因的sh RNA,插入含绿色荧光蛋白(green fluorescent protein,GFP)编码基因的p Magic 7.1载体,构建p21/Waf1-sh RNA重组慢病毒质粒p LVT1051,并进行PCR和测序鉴定。将p LVT1051、p CMV-VSV-G和p CMV-d R8.91三种质粒共转染293T细胞,包装产生慢病毒。将携带有p21/Waf1-sh RNA的慢病毒感染EJ细胞,采用嘌呤霉素筛选p21/Waf1-sh RNA稳定转入的EJ细胞,采用实时荧光定量PCR和蛋白质印迹法分别检测p21/Waf1-sh RNA稳定感染后EJ细胞中p21/waf1 m RNA和蛋白的表达水平。将膀胱癌特异性溶瘤腺病毒Ad/PSCAE/UPⅡ/E1A感染p 21/waf 1基因沉默的EJ细胞,采用蛋白质印迹法检测病毒复制相关指标E1A和Hexon蛋白的表达水平,MTT法检测腺病毒Ad/PSCAE/UPⅡ/E1A对EJ细胞增殖的影响。结果 :成功构建了携带有p21/Waf1-sh RNA的重组慢病毒表达载体并获得相应的慢病毒;经嘌呤霉素筛选2周后成功获得稳定感染的EJ细胞。p21/Waf1-sh RNA能明显降低EJ细胞中p21/Waf1 m RNA及蛋白的表达水平,差异均有统计学意义(P值均<0.05)。膀胱癌特异性溶瘤腺病毒(Ad/PSCAE/UPⅡ/E1A)作用于p 21/waf 1基因沉默的EJ细胞后,其病毒复制相关指标E1A和Hexon的表达水平明显上调,对EJ细胞的增殖抑制作用明显增强(P<0.01)。结论 :成功建立了p21/Waf1基因沉默表达的EJ细胞,沉默p21/Waf1基因的表达能增强膀胱癌特异性溶瘤腺病毒的复制能力,并对EJ细胞的增殖有明显的抑制作用。Objective： To investigate the effects of p21/Waf1 gene silencing on replicative potential of bladder cancer-specific oncolytic adenovirus and the proliferation inhibition of EJ cells. Methods： The shRNA targeting p21/Waf1 gene （p21/Waf1-shNA） was designed and synthesized, then the annealing oligonucleotide fragments were subcloned into pMagic 7.1 vector containing coding gene of green fluorescent protein （GFP） to construct recombinant lentiviral plasmid pLVT1051, which was confirmed by PCR and DNA sequencing. The 293T cells were co-transfected with three plasmids including pLVT1051, pCMV-VSV-G and pCMV- dR8.91 to produce the recombinant lentivirus. The EJ cells were infected with recombinant lentivirus carrying p21/Waf1-shRNA, and the puromycin was used to screen out the stably infected EJ cells. The expression levels of p21/Waf1 mRNA and protein in EJ cells after infection with recombinant lentivirus carrying p21/Waf1-shRNA were detected by real-time fluorescent quantitative-PCR and Western blotting, respectively. The p21/Waf1 gene-silenced EJ cells were infected with bladder cancer-specific oncolytic adenovirus Ad/PSCAE/UPII/E1A, then the expression levels of virus replication-related proteins E1A and Hexon were detected by Western blotting, and the cytotoxic effect on EJ cells was examined by MTT method. Results： Recombinant lentiviral vector carrying p21/Waf1 -shRNA targeting p21 /Wall gene was successfully established and confirmed by DNA sequencing. The recombinant lentivirus was harvested. The stably infected EJ cells were successfully screened out by using puromycin forr two weeks. The expression levels of p21/Waf1 mRNA and protein in EJ cells infected with recombinant lentivirus carrying p21/Waf1-shRNA were significantly reduced （both P 〈 0.05）. The expression levels of E1A and Hexon proteins in p21/Waf1 gene-silenced EJ cells infected with bladder cancer-specific oncolytic adenovirus Ad/PSCAE/UPII/E1A were up-regulated, and the proliferation inhibition of EJ cells was enhanced （P �

关 键 词：膀胱肿瘤 RNA 小分子干扰 慢病毒感染 p21/Waf1基因 病毒复制 EJ细胞 

分 类 号：R737.14[医药卫生—肿瘤]