靶向survivin基因微小RNA真核表达载体的构建及其对人结肠癌细胞增殖的影响  

Construction and inhibitory effect of microRNA expression vector targeting survivin on proliferation of human colorectal carcinoma HT-29 cells

作　　者：崔运福 郝涛[1] 王荣华[1] 李宝松[1] 马冲[1] 樊鹏[1] 

机构地区：[1]滨州医学院附属医院肛肠外科,256603

出　　处：《国际肿瘤学杂志》2015年第1期22-26,共5页Journal of International Oncology

摘　　要：目的 构建靶向survivin基因的微小RNA（miRNA）真核表达载体,探讨其对人结肠癌细胞（HT-29）增殖、凋亡的影响.方法 设计合成针对survivin基因的miRNA序列,定向克隆至pcDNATM6.2-GW/Em GFP-miR真核表达载体上,构建靶向survivin的miRNA重组质粒.HT-29细胞接种于6孔板,分为空白对照组、转染试剂组、空质粒组（阴性对照组）以及目的基因组（阳性对照组）4组.瞬时转染后收集各组细胞,应用流式细胞仪检测各组细胞的增殖指数及凋亡率,实时荧光定量聚合酶链反应和免疫印迹法（Western blot）检测靶基因（survivin） mRNA和蛋白的表达.结果 HT-29细胞转染后,目的基因组增殖指数与正常组、转染试剂组、空质粒组相比明显降低（17.98％±2.35％ vs 38.04％±2.11％ vs36.73％ ±2.51％ vs36.57％±3.05％;t=20.05,P ＜0.01;t =18.75,P＜0.01;t=18.59,P＜0.01）,凋亡率明显增高（19.54％±1.74％ vs 3.13％±0.29％ vs 3.70％ ±0.44％ vs 3.61％±0.50％; t=16.40,P＜0.01;t=15.84,P＜0.01;t=15.92,P＜0.01）.目的基因组survivin mRNA与其他各组相比明显降低（=0.68,P＜0.01;=0.58,P＜0.01;t =0.61,P＜0.01）,蛋白的表达亦明显降低（t=0.64,P＜0.01;t =0.62,P＜0.01;t =0.67,P＜0.01）.结论 靶向沉默结肠癌HT-29细胞的survivin基因可以明显抑制细胞增殖,并诱导细胞凋亡.Objective To construct microRNA （miRNA） expression vector targeting surviving,and to investigate its effect on transfected human colorectal carcinoma （HT-29） cell apoptosis and proliferation.Methods miRNA targeting survivin was synthesized and transfected HT-29 cells by lipofectin.HT-29 cells were cultured in the 6 orifices.The cultured cells were divided into control,liposome,negative control and positive control groups.Transient transfected cells were collected and the proliferation index and apoptosis rate of HT-29 cells were detected by flow cytometry.The expressions of survivin mRNA and protein were detected by RT-PCR and Western blot.Results The proliferation index and apoptosis rate of the positive control group were significantly higher compared with normal group,transfection group and mock-vehicle group （17.98％ ± 2.35％ vs 38.04％ ±2.11％ vs 36.73％ ±2.51％ vs 36.57％ ±3.05％; t =20.05,P＜0.01; t =18.75,P＜0.01; t=18.59,P＜0.01; 19.54％ ±1.74％ vs 3.13％ ±0.29％ vs 3.70％ ±0.44％ vs 3.61％ ± 0.50％ ; t =16.40,P ＜ 0.01 ; t =15.84,P ＜ 0.01 ; t =15.92,P ＜ 0.01）.Survivin mRNA and protein expression levels were specifically suppressed in transfected HT-29 cells （t =0.68,P ＜0.01 ; t =0.58,P ＜ 0.01; t=0.61,P＜0.01;t=0.64,P＜0.01; t=0.62,P＜0.01;t=0.67,P＜0.01）.Conclusion Survivin targeted silence can effectively decrease the expression of survivin mRNA and protein,induce colorectal carcinoma HT-29 cell apoptosis and inhibit cell proliferation.

关 键 词：微RNAS 结肠肿瘤 细胞增殖 SURVIVIN 

分 类 号：R735.35[医药卫生—肿瘤]