RNA干扰Ezrin体外对肾癌细胞株786-0增殖、凋亡的影响(英文)  

Effects of Ezrin on the Proliferation and Apoptosis of Renal Cell Carcinoma

作　　者：杨旭凯[1] 郭秀全[2] 王养民[1] 周逢海[1] 蓝天[1] 乔够梅[1] 

机构地区：[1]兰州军区兰州总医院全军泌尿外科中心,甘肃兰州730050 [2]兰州大学第二临床医学院泌尿外科,甘肃兰州730000

出　　处：《现代生物医学进展》2014年第25期4811-4815,共5页Progress in Modern Biomedicine

基　　金：Comprehensive prevention research of urinary calculi on the northwest troops(CLZ12J004)~~

摘　　要：目的:构建膜-细胞骨架联接蛋白Ezrin基因特异性短发卡RAN表达载体(small hairpin RNA,shRNA),探讨其对肾癌细胞株786-0细胞凋亡、增殖的影响。方法:以Ezrin为靶基因,以Pgenesil-1质粒为载体,设计和构建重组体,设计2条发夹式RNA(shRNA),合成后克隆入载体Pgenesil-1,扩增并中量提取质粒,应用脂质体Lipofectamine 2000转染进786-0细胞,重组质粒转染786-0肾癌细胞株,用运实时荧光定量PCR进行筛选鉴定,筛选出抑制率较高的重组质粒载体shRNA-Ezrin1,用shRNA-Ezrin1转染786-0细胞,采用MTT、流式细胞仪、电镜检测,观察RNA干扰Ezrin后肾癌细胞株786-0细胞增殖能力的改变。结果:shRNA干扰后786-0细胞增殖活性减弱,G0/G1时段明显延长(P<0.01),PI缩短(P<0.01),细胞凋亡率增加(P<0.01)。结论:Ezrin与肾癌细胞凋亡、增殖有关,有望成为肾癌基因治疗的一个新靶点。Objective： To construct the short hairpin RAN which is a specificity expression vector of membrane-cell skeleton join Ezrin protein gene, and to investigat its roles in proliferation and apoptosis of RCC （Renal Cell Carcinoma）. Methods： Ezrin was designed, chemically synthesized and inserted into plasmid pGenesil-shRNA, which was transformed into DH5o~. The recombinant plasmid was extracted in middle quantity and transferred into renal carcinoma cell line 786-0 by Lipofectamine 2000. Ezrin expressing in 786-0 cells transfected by shRNA recombinant plasmid was detected by qRT-PCR. 786-0 cells were transferred by effective shRNA plasmid and cultured in 1640 media containing G418 （800 Ixg/ml）. The blocking effect of shRNA-Ezrin was detected by qRT-PCR; The pro- liferation and apoptosis were assessed by 3-（4, 5-dimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide （MTT） assay and Flow cytometry. Protein expression was assayed by western blotting. Autophagy was evaluated by transmission electron microscopy. Results： After transfected with pGeneSil-l-Ezrin, the proliferation capability of 786-0 cells decreased dramatically; Compared with that in the cells treated with shRNA-Ezrinl, the percentage of cells in G0/G1 stage were significantly higher than those in untransfected cells and 786-0cells transferred with shRNA-HK（P〈0.01 ）and PI（proliferation index） decreased in the cells treated with shRNA-Ezrinl compared with those in untransferred cells and 786-0 cells transferred with shRNA-HK （P〈0.01）. Conclusions： Ezrin plays an important role in cell prolifera- tion and apoptosis of human renal carcinoma cells, which may be regarded as a promising target for tumor gene therapy.

关 键 词：EZRIN蛋白 肾癌 RNA干扰 

分 类 号：R737.11[医药卫生—肿瘤]