Wilms瘤基因1低表达和过表达乳腺癌细胞模型的建立  

Establishment of the Wilms' tumor gene 1 low and over-expression models in breast cancer cells

作　　者：齐晓伟[1] 杨新华[1] 张毅[1] 范林军[1] 张帆[1] 陈莉[1] 唐振宁[1] 张彦[1] 杨晓宁[1] 宗贝歌[1] 胡保全[1] 王明浩[1] 姜军[1] 

机构地区：[1]第三军医大学西南医院乳腺外科,重庆400038

出　　处：《中华乳腺病杂志（电子版）》2013年第4期1-6,共6页Chinese Journal of Breast Disease(Electronic Edition)

基　　金：国家自然科学基金资助项目(81102030)

摘　　要：目的应用RNA干扰(RNAi)和RNA激活(RNAa)技术,分别构建小干扰RNA(siRNA)和双链RNA(dsRNA),建立Wilms瘤基因1(WT1)低表达和过表达的乳腺癌细胞模型。方法以国外学者提供的3条序列(siRNA-516、siRNA-803、siRNA-1029)构建WT1 siRNA干扰表达WT1,以研究已证实可上调WT1表达的序列(dsRNA-319)构建dsRNA过表达WT1,通过脂质体(LipofectamineTM2000)分别将siRNA和dsRNA转入MDA-MB-321和MCF-7细胞。WT1有效siRNA筛选实验分为6组:WT1siRNA-516、WT1 siRNA-803、WT1 siRNA-1029、阴性对照、脂质体组和空白细胞组;观察转染效率的时间点为转染后24、48、72 h。WT1 dsRNA的筛选实验分为3组:WT1 dsRNA-319、阴性对照组和空白细胞组;观察转染效率的时间点为转染后48、72、96 h。通过实时定量PCR(qRT-PCR)和Western Blotting筛选作用效果最明显的siRNA和dsRNA。使用单因素方差分析进行统计学分析。结果成功构建WT1siRNA-516、WT1 siRNA-803和WT1 siRNA-1029共3个siRNA,并转染到MDA-MB-231细胞中,转染效率达90%以上。上述3个WT1 siRNA均能够抑制WT1 mRNA和蛋白的表达,以转染后48 h WT1 siRNA-1029的效果最为显著[WT1 siRNA-1029组WT1 mRNA表达水平与空白细胞组相比显著降低(0.49±0.02比1.00±0.08,P=0.00),其蛋白表达水平也明显降低]。成功将WT1 dsRNA-319转染到MCF-7细胞中,转染效率达90%以上。50μmol/L的WT1 dsRNA-319转染后96 h,MCF-7细胞的WT1过表达最为显著[WT1 dsRNA-319组的WT1 mRNA表达水平与空白细胞组相比显著升高(319.06±14.84比1.00±0.07,P=0.00),其蛋白表达水平也明显升高]。结论成功建立低表达WT1的MDA-MB-231细胞和过表达WT1的MCF-7细胞模型,为后续进一步研究WT1在乳腺癌中的生物学行为奠定了基础。Objective To construct small interfering RNA （siRNA） and double strands RNA （dsRNA） by RNA interference （RNA interference,RNAi） and RNA activation （RNA activating,RNAa）,and establish the low and over-expression model of Wilms＇ tumor gene 1 （WT1） in breast cancer cells.Methods Three sequences （siRNA-516,siRNA-803 and siRNA-1029） established by foreign scholars were adopted to construct WT1 siRNA; one sequence （dsRNA-319）which demonstrated to be able to up-regulate WT1 expression was adopted to construct WT1 dsRNA.siRNA and dsRNA were transfected into MDA-MB-321 and MCF-7 cells by LipofectamineTM 2000,respectively.WT1 siRNA screening experiment contained six groups：WT1 siRNA-516,WT1 siRNA-803,WT1 siRNA-1029,negative control,transfection reagent and blank groups,and the points of time for observation were at 24,48 and 72 h after transfection,respectively.WT1 dsRNA screening experiments contained three groups：WT1 dsRNA-319,negative control and blank group,and the point of time for observation was at 48,72 and 96 h after transfection respectively.Quantitative real-time PCR（qRT-PCR） and Western Blotting were performed to select siRNA and dsRNA with obvious impacts on WT1 expression.The one-way ANOVA was used for statistical analysis.Results Three WT1 siRNAs （WT1 siRNA-516,WT1 siRNA-803 and WT1 siRNA-1029） were successfully constructed and transfected into MDA-MB-231 cells with transfection efficiency ＞90％.WT1 siRNAs could effectively inhibit the expression of WT1 mRNA and protein,among which siRNA-1029 could inhibit the WT1 mRNA expression at 48 h after transfection most significantly （0.49±0.02 for WT1 siRNA-1029 group vs 1.00±0.08 for blank group,P=0.00;the protein expression was also decreased dramatically）.WT1 dsRNA-319 could increase the expression of WT1 mRNA in MCF-7 cell with transfection efficiency ＞90％.The most significant impact was achieved in 50 μmol/L WT1 dsRNA-319 group at 96 h after transfection （319.06±14.84 for WT1 dsRNA-319 group vs 1.00±0.

关 键 词：Wilms瘤基因1 RNA干扰 RNA激活 乳腺肿瘤 

分 类 号：R737.9[医药卫生—肿瘤]