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-stranded RNA (dsRNA) using RNA interference (RNAi) and RNAi (RNAi) technology and establish a model of breast cancer cell line with low and overexpression of Wilms tumor gene 1 (WT1) Methods WT1 siRNA was constructed by interfering with WT1 siRNA targeting three sequences (siRNA-516, siRNA-803, siRNA-1029) to study the expression of WT1 over dsRNA-319 SiRNA and dsRNA were transfected into MDA-MB-321 and MCF-7 cells, respectively, by lipofectamine (TM) 2000. WT1 siRNA screening experiments were divided into 6 groups: WT1 siRNA-516, WT1 siRNA-803, WT1 siRNA-1029, negative control group, liposome group and blank group. The time point of transfection efficiency was 24,48 , 72 h. WT1 dsRNA-319, negative control group and blank group were divided into three groups. The transfection efficiency was observed at 48, 72 and 96 h after transfection. The most potent siRNAs and dsRNAs were screened by real-time quantitative PCR (qRT-PCR) and Western Blotting. One-way analysis of variance was used for statistical analysis. Results A total of 3 siRNAs of WT1 siRNA-516, WT1 siRNA-803 and WT1 siRNA-1029 were successfully constructed and transfected into MDA-MB-231 cells. The transfection efficiency was over 90%. The three WT1 siRNAs both inhibited the expression of WT1 mRNA and protein, and WT1 siRNA-1029 had the most significant effect at 48 h after transfection [WT1 siRNA-1029 showed a significant decrease in WT1 mRNA expression compared with the blank group (0.49 ± 0.02 vs 1.00 ± 0.08, P = 0.00), and its protein expression level was also significantly reduced]. Transfection of WT1 dsRNA-319 into MCF-7 cells was successful and the transfection efficiency was over 90%. WT1 dsRNA-319 transfected with 50μmol / L WT1 dsRNA-319 96h, WT1 overexpression of MCF-7 cells most significant [WT1 dsRNA-319 WT1 mRNA expression levels were significantly higher than the blank group (319.06 ± 14.84 1.00 ± 0.07, P = 0.00), the protein expression level was also significantly higher]. Conclusion The successful establishment of MDA-MB-231 cells with low expression of WT1 and MCF-7 cells overexpressing WT1 lay a foundation for further study on the biological behavior of WT1 in breast cancer.