NEDD9基因miRNA干扰质粒的构建及鉴定  

Construction and identification of miRNA recombinant eukaryotic expression vectors of NEDD9

作　　者：李羚[1] 赵金朋[1] 冯锦[1] 谢海彬[1] 尹勇[1] 吴雨岗[1] 李忠[1] 

机构地区：[1]常州市第一人民医院(苏州大学附属第三医院)胃肠外科,江苏省213000

出　　处：《江苏医药》2016年第2期133-136,F0002,共5页Jiangsu Medical Journal

基　　金：常州市卫生局重大课题(ZD200906);常州市科技局项目(CJ20140047)

摘　　要：目的构建神经元表达发育下调基因9(NEDD9)特异性微小核糖核酸(miRNA)干扰质粒,检测其在胃癌SGC-7901细胞中的干扰效果。方法根据GenBank中提供的NEDD9基因序列,设计4种NEDD9靶向干扰序列(MR0112-1、MR0112-2、MR0112-3和MR0112-4)及1种阴性对照干扰序列(MR0112-N),连接到pcDNA6.2-GW/EmGFP-miR载体中,构建重组质粒。重组质粒转染人胃癌细胞株SGC-7901;采用FQ-PCR和Western blot法检测转染前后SGC-7901细胞中NEDD9mRNA及蛋白的表达。结果测序表明,4种干扰质粒的干扰序列及读框完全正确。FQ-PCR和Western blot结果显示,转染MR0112-1、MR0112-2、MR0112-3和MR0112-4质粒后其NEDD9表达较未处理组分别下降88.01%、96.70%、78.55%和81.34%(P<0.05),以MR0112-2干扰效果最为明显。阴性质粒组NEDD9mRNA表达与未处理组比较无明显变化(P>0.05)。结论成功构建了NEDD9干扰质粒,为进一步研究NEDD9基因表达在胃癌增殖、迁移、侵袭过程中的作用提供实验基础。Objective To construct the microRNA（miRNA）eukaryotic expression vectors of neural precursor cell expressed,developmentally down-regulated 9（NEDD9）and explore its activity of inhibiting the mRNA and protein expressions of NEDD9 in human gastric cancer SGC-7901 cells.Methods Four miRNA interference sequences specific to NEDD9（MR0112-1,MR0112-2,MR0112-3and MR0112-4）and one non-homologous negative control sequence（MR0112-N）were designed and constructed.The miRNA sequences and the control were inserted into pcDNA6.2-GW/EmGFP-miR vector.After identification,restriction analysis and DNA sequencing were conducted.The recombinant miRNA vectors were transfected into SGC-7901 cells.The expressions of NEDD9 mRNA and protein were detected by FQ-PCR and Western blot,respectively.Results DNA sequencing showed that eukaryotic expression vectors targeting NEDD9 gene possessed correct read frame and nucleotide sequence.FQ-PCR and Western blot results showed that compared to the controls,the levels of NEDD9 mRNA and protein in transfected SGC-7901 cells significantly reduced by 88.01%,96.70%,78.55% and 81.34% for expression vectors of MR0112-1,MR0112-2,MR0112-3and MR0112-4,respectively.Conclusion The miRNA eukaryotic expression vectors targeting NEDD9 are constructed successfully,which may provide a foundation for the study of NEDD9 gene function in gastric carcinoma.

关 键 词：神经元表达发育下调基因9 胃癌 微小核糖核酸 

分 类 号：R735[医药卫生—肿瘤]