| 杨旭凯 郭秀全 王养民 周逢海 蓝天 乔够梅.RNA干扰Ezrin体外对肾癌细胞株786-0增殖、凋亡的影响[J].,2014,14(25):4811-4815 |
| RNA干扰Ezrin体外对肾癌细胞株786-0增殖、凋亡的影响 |
| Effects of Ezrin on the Proliferation and Apoptosis of Renal Cell Carcinoma |
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| DOI： |
| 中文关键词: Ezrin 蛋白 肾癌 RNA干扰 |
| 英文关键词: Ezrin Renal cell carcinoma RNA interference |
| 基金项目:西北地区官兵泌尿系结石防治的综合研究（CLZ12J004） |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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 DH5alpha. 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 ug/ml). The blocking effect of shRNA-Ezrin was detected by qRT-PCR; The proliferation
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-Ezrin1, 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-Ezrin1 compared with those
in untransferred cells and 786-0 cells transferred with shRNA-HK（P<0.01）.Conclusion:Ezrin plays an important role in cell proliferation
and apoptosis of human renal carcinoma cells, which may be regarded as a promising target for tumor gene therapy. |
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