| 吕佳孙晓阳△ 丁涟沭金孝东卜向飞卞爱苗蒋健.EZH2 靶向的shRNA 质粒的构建和有效靶序列的筛选*[J].,2012,12(7):1211-1214 |
| EZH2 靶向的shRNA 质粒的构建和有效靶序列的筛选* |
| Construction and Screening of shRNA-Expressing PlasmidTargeting to EZH2 Gene |
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| DOI： |
| 中文关键词: zeste 基因增强子人类同源物2 RNA 干扰 质粒 胶质瘤 |
| 英文关键词: EZH2 RNAi Plasmid Glioma |
| 基金项目:江苏省医学重点人才基金RC2007029；江苏省社会发展项目BS2007037；
淮安市科技发展基金HAS07025；南京医科大学科技发展基金重点项目06NMUZ047 |
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| 中文摘要: |
| 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 hairpin
RNA，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。 |
| 英文摘要: |
| 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. |
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