人Hiwi miRNA干扰质粒的构建及其鉴定  

Construction and identification of miRNA recombinant eukaryotic expression vectors of Hiwi

作　　者：郝波[1] 蒋建霞[1] 施瑞华[1] 张国新[1] 成道祥 

机构地区：[1]南京医科大学第一附属医院消化科,江苏南京210029 [2]江苏省陆军预备役高射炮兵第一师第一团,江苏南京210029

出　　处：《实用临床医药杂志》2013年第3期1-4,27,共5页Journal of Clinical Medicine in Practice

基　　金：国家自然基金项目(NSFC81101800);江苏省南京市科技发展研究项目资助(2010ZD220)

摘　　要：目的构建人Hiwi基因miRNA干扰质粒,探讨其对肝癌7721细胞株中Hiwi基因和蛋白表达的阻抑作用。方法根据Homo sapiens Hiwi基因序列,设计RNA干扰靶点,构建4对Hiwi的pcDNATM6.2-GW/EmGFPmiR miRNA及1对无效对照miRNA干扰质粒并将其转染至肝癌7721细胞中,通过RT-PCR和Western blot检测7721细胞中HiwimRNA和蛋白的表达水平。结果成功构建了4对Hiwi miRNA干扰质粒及无效干扰质粒,测序表明Hiwi干扰序列及读框完全正确。RT-PCR和Western blot结果显示,与未转染组及阴性对照组比较,转染该载体能有效下调7721细胞株中mRNA和蛋白的表达。结论成功构建了Hiwi的干扰表达载体,为进一步探讨Hiwi基因在肝癌中的相关机制提供了实验基础。Objective To construct the miRNA eukaryotic expression vectors of HIWI, and explore its activity of inhibiting the gene and protein expression in human Hepatocellular carcinoma 7721 cell. Methods Four specific micro interference RNA expression vectors specific to Hiwi and one non-homologous negative control vector were designed and constructed. After identification, restriction analysis and DNA sequencing were conducted, the miRNA vectors and the control one were inserted into pcDNA TM6.2-GW/EmGFPmiR vector. The recombinant miRNA vectors were transfected into 7721 cell. The expression of Hiwi gene miRNA was determined by RT-PCR and Western blot was used to examine the expression of target protein. Results Sequencing suggested that eukaryotic expression vectors targeting Hiwi gene possessed correct read frame and nucleotide sequence. RT-PCR and Western blot results showed that the level of mRNA and target protein of Hiwi in transfected 7721 cells significantly reduced compared to the controls. Conclusion MiRNA eukaryotic expression vectors targeting Hiwi are constructed successfully, which will provide the foundation for study of Hiwi gene function in hepatocellular carcinoma.

关 键 词：Hiwi基因 miRNA干扰 真核表达质粒 肝癌 

分 类 号：R735.7[医药卫生—肿瘤]