EZH2 靶向的shRNA 质粒的构建和有效靶序列的筛选*

目的:构建并筛选靶向zeste基因增强子人类同源物2(Enhancer of zeste homologue 2,EZH2)的短发卡RNA(short hairpinRNA,shRNA)的质粒表达载体。方法:设计并合成针对EZH2基因的带有小发夹结构的DNA片段并克隆至质粒pGPU6/GFP/Neo中,经酶切和测序分析后转染入胶质瘤U251细胞,分别应用实时荧光定量PCR和Western bloting在mRNA和蛋白质水平观察其对EZH2基因表达的沉默效果。结果:重组质粒构建成功,并成功转染入胶质瘤U251细胞,转染效率大约为70%。其中,以靶向hEZH2-715序列的质粒抑制效果最好,其对U251细胞EZH2 mRNA和protein抑制率分别为55%和89%。结论:成功构建了能高效抑制EZH2基因表达shRNA的重组质粒,为下一步探索EZH2在胶质瘤细胞中的生物学作用奠定了基础。

Construction and Screening of shRNA-Expressing Plasmid Targeting to EZH2 Gene

Objective: To construct and screen shRNA-expressing plasimd targeting with EZH2 gene.Methods: The DNA oligonucleotide fragments targeting to human EZH2 gene were designed and synthesized,then they were cloned into pGPU6/GFP/Neo plasmid.The recombinant plasmids were identified by restriction enzyme and sequencing analyses,then were transfected into U251 glioma cells;The transfection efficiency was observed and the EZH2 gene silencing effect was detected by quantitative RT-PCR and Western bloting.Results:The recombinant plasmids were constructed and transfected into U251 glioma cells successfully.The transfection rate was approximately 70%.Among them,the inhibition efficiency of the reconbinant plasimd targeting to hEZH2-715 sequence was the best.It’s inhibition rates were 55% and 89% in the level of EZH2 mRNA and protein in the U251 cells,respectively.Conclusion: The recombinant plasimd expressing EZH2-shRNA that could suppress EZH2 gene’s expression effectively was constructed successfully,which laid the foundation for next step to investigate EZH2 gene’s biological role in glioma cells.