抑制EZH2基因表达的shRNA载体的构建及其作用  

Construction and effect of shRNA vector silencing EZH2 expression

作　　者：吕艳锋[1] 韩冰冰[2] 禹化龙[1] 王建新[1] 

机构地区：[1]山东大学第二医院肛肠外科,济南250033 [2]山东中医药大学微循环实验室,济南250014

出　　处：《山东大学学报（医学版）》2013年第9期31-34,共4页Journal of Shandong University:Health Sciences

基　　金：山东省自然科学基金(ZR2011HQ049);济南市青年科技明星计划(20090208;201004055)

摘　　要：目的构建zeste基因增强子同源物2(EZH2)靶向的小发夹RNA(shRNA)重组表达载体,探讨抑制EZH2基因表达后其在结直肠癌细胞中的作用。方法根据EZH2cDNA序列设计具有短发夹结构的两条DNA序列,与载体pGFP-V-RS构建重组表达载体,鉴定后转染至SW480细胞。将其随机分为阴性对照组和基因沉默组,RTPCR和蛋白质印迹法检测抑制效果,MTT法检测细胞增殖情况。结果成功构建了抑制EZH2基因表达的干扰质粒。阴性对照组EZH2 mRNA的表达是基因沉默组的5.8倍(P<0.01),基因沉默组EZH2的蛋白明显低于阴性对照组(P<0.05)。与阴性对照组相比,基因沉默组细胞生长受到明显抑制(P<0.05)。结论 EZH2靶向shRNA重组表达载体构建成功,并能显著抑制EZH2基因的表达,为进一步研究EZH2基因在肿瘤中的作用机制提供了基础。Objective To construct a recombinant short hairpin RNA （shRNA） expression vector targeting enhancer of zeste homolog 2 （EZH2） gene and explore its effect on colorectal cancer cells. Methods Two DNA sequences with short hairpin structure were designed according to the EZH2 cDNA sequence and cloned into pGFP-V-RS vector to con- struct a recombinant expression vector silencing EZH2 gene, After identification, the shRNA-expressing vector was then transfected into SW480 cells. Then transfected SW480 cells were divided into negative control group and gene silencing group. RT-PCR and Western blotting were used to detect inhibitory effect. MTF was used to detect cell proliferation. Results A recombinant vector harboring shRNA of EZH2 was successfully constructed in this work. With respect to as- sessment of interference efficiency, the expression of EZH2 mRNA in negative control group was 5.8 times （P 〈 0.01 ） of that in gene silencing group. And the expression of EZH2 protein in gene silencing group was significantly lower than that in negative control group （ P 〈 0.05 ）. Moreover, the cell viability in gene silencing group was significantly reduced than that in negative control group （P 〈 0.05）. Conclusion The present study demonstrates that a recombinant shRNA expression vector targeting EZH2 gene was successfully constructed, with significant inhibitory effect on proliferation of SW480 cells. This lays an experimental foundation for further exploring the mechanism underlying the action of EZH2 gene on tumor biology.

关 键 词：EZH2基因 RNA干扰 SW480细胞 蛋白质印迹法 MTT法 

分 类 号：R735.3[医药卫生—肿瘤]