乙酰肝素酶基因靶向特异性RNA干扰重组表达载体的构建及筛选  

Construction and screening of specific short hairpin RNA vector targeting heparanase gene

作　　者：刘阳[1] 霍强[1] 许立薇[1] 蒋志文[1] 

机构地区：[1]蚌埠医学院药学系,安徽省生化药物工程技术研究中心,安徽蚌埠233030

出　　处：《南方医科大学学报》2010年第10期2340-2342,2346,共4页Journal of Southern Medical University

基　　金：安徽省高等学校省级自然科学研究项目(KJ2009B150Z)~~

摘　　要：目的构建对人乙酰肝素酶(HPA)基因有特异性抑制作用的短发夹状RNA表达载体。方法根据GenBank数据库提供的人HPA基因核苷酸序列,按照shRNA设计原则设计5对特异性的寡核苷酸序列及阴性对照,分别克隆到pGPU6/GFP/Neo载体中,酶切鉴定和测序验证阳性克隆。用脂质体转染入MDA-MB-231细胞,分别以荧光定量PCR、Western blot分析其对HPA在mRNA水平和蛋白质水平上表达的影响。结果经酶切、测序鉴定均证实重组质粒构建成功。在构建的5个载体中,HPSE-1组及HPSE-5组中MDA-MB-231细胞HPA基因mRNA和蛋白质表达水平明显下降,而对照组未发生明显改变。结论成功构建了2个靶向人HPA的shRNA表达载体,转染MDA-MB-231细胞后可高效抑制HPA基因表达,为进一步研究HPA的表达与乳腺癌迁移和侵袭的机制奠定了基础。Objective To construct short hairpin RNA（shRNA） expression vectors of RNA for specific silencing of heparanase（HPA） gene.Methods The genomic sequence of HPA gene was retrieved from GenBank and the cDNA encoding shRNA for HPA gene silencing was designed.Five specific interference sequences and a random negative control sequence were inserted into the vectorpGPU6/GFP/Neo.Afterverification by restriction enzyme digestion and sequence analysis,the recombinant vectors were transfected into MDA-MB-231 cells via lipofectamin.Fluorescent quantitative PCR and Western blotting were employed to detect the expression of HPA gene expressions in the transfected cells at the mRNA and protein levels,respectively.Results Both restriction analysis and sequencing confirmed correct construction of the shRNA vectors.Transfected with the specific siRNA vectors HPSE-1 and HPSE-5 resulted in significantly decreased expression level of HPA protein in MDA-MB-231 cells,while negative control vector produced no significant changes in HPA expressions.Conclusion We have obtained two shRNA vectors which can significantly down-regulate HPA expressions in MDA-MB-231 cells,which facilitates further investigation of the role HPA may play in the invasiveness and metastasis of human breast cancer.

关 键 词：乙酰肝素酶 RNA干扰 MDA—MB-231细胞 

分 类 号：R730.2[医药卫生—肿瘤]