宫颈癌中乙酰肝素酶RNA干扰特异性靶点的构建及筛选  

Construction and screening of the heparanase RNA interference specific targets in cervical cancer

作　　者：吕琼莹[1] 张蔚[1] 程静[1] 张文婷[1] 钟亚娟[1] 

机构地区：[1]武汉大学人民医院妇一科,武汉430060

出　　处：《中华实用诊断与治疗杂志》2013年第8期741-743,共3页Journal of Chinese Practical Diagnosis and Therapy

基　　金：国家自然科学基金(31170329)

摘　　要：目的设计合成对人乙酰肝素酶(heparanase,HPA)基因有特异性抑制作用的短发夹状RNA表达载体,筛选出对HPA沉默效果最佳的质粒。方法根据GenBank数据库提供的人HPA基因核苷酸序列,按短发夹RNA设计原则设计4对特异性寡核苷酸序列及阴性对照,分别克隆到pYr-1.1质粒载体中。用脂质体转染入Hela细胞,分别采用RT-PCR与细胞免疫荧光分析其对HPA在mRNA和蛋白质水平上表达的影响。结果经测序鉴定证实重组质粒构建成功;在构建的4个载体中,HPA-592组Hela细胞和Caski细胞HPA基因mRNA和蛋白质表达水平明显下降,对照组未发生明显改变。结论成功筛选出靶向人HPA的shRNA表达载体,转染宫颈癌Hela细胞和Caski细胞后可高效抑制HPA基因表达。Objective To design and synthesize short hairpin RNA （shRNA） expression vector of RNA for specific silencing of heparanase （HPA） gene, and screen the plasmid with the best silence effect on HPA. Methods The genomie sequence of HPA gene was retrieved from GenBank and the cDNA encoding shRNA for HPA gene silencing was designed. Four specific interference sequences and a random negative control sequence were inserted into the vector pYr- 1. 1. Vectors were transfeeted into Hela and Caski cells via lipofectamin. Real-time PCR technique and immunofluorescenee method were employed to detect HPA gene expressions in the transfected cells at the mRNA and protein levels, respectively. Results Sequencing confirmed correct construction of the shRNA vectors. Transfected with the specific shRNA vectors HPA-592 resulted in significantly decreased expression level of HPA protein in Hela and Caski cells, while negative control vector produced no significant changes in HPA expressions. Conclusion The successfully obtained shRNA expression vector of HPA can effectively inhibit the expression of HPA gene in transfected Hela and Caski cells in cervical cancer.

关 键 词：宫颈癌 HPA HELA细胞 CASKI细胞 RNAI 

分 类 号：R737.33[医药卫生—肿瘤]