EZH2 shRNA真核表达载体的构建及其在卵巢癌A2780细胞系的稳定表达

Construction of EZH2 shRNA Eukaryotic Expression Vector and Its Stable Expression in A2780 Cells

目的采用基因工程技术,构建EZH2 shRNA的真核表达载体,获得稳定表达干扰质粒的卵巢癌A2780细胞系。方法合成特异性干扰EZH2的shRNA片段并定向克隆插入Supersilencing shRNA质粒表达载体pGPU6/GFP/Neo,获得表达载体pGPU6/GFP/Neo-shEZH2。脂质体介导重组质粒转染A2780细胞,经G418加压筛选获得稳定转染细胞株,Real time RT-PCR和Western blot检测转染后细胞系EZH2的表达。结果测序结果证实重组载体构建成功,稳定转染shEZH2的A2780细胞EZH2的mRNA和蛋白水平下降(90.20±1.66)%和(73.80±5.49)%,P<0.01,差异有统计学意义。结论成功构建EZH2 shRNA表达载体,获得EZH2基因稳定沉默的A2780细胞系,为进一步研究以EZH2为靶点的卵巢癌治疗奠定实验基础。

Objective To construct short hair RNA（shRNA）eukaryotic expression vector targeting EZH2 and establish the stable EZH2-knockdown A2780 cell line by gene engineering technology.Methods The shRNA sequences targeting EZH2 gene were synthesized and inserted into Supersilencing shRNA plasmid expression vector pGPU6/GFP/Neo,and the positive clone was named pGPU6/GFP/Neo-shEZH2.The recombinant vector was transfected into A2780 cells by LipofectamineTM 2000,and the stably transfected cells were obtained after being screened with G418.The mRNA and protein levels of EZH2 in stably transfected cells were detected by real-time RT-PCR and Western blot.Results The shRNA expression vector was confirmed by DNA sequencing.The expression of EZH2 mRNA and protein was decreased by（90.20±1.66）% and（73.80±5.49）% respectively in shEZH2-transfected cells as compared with vector-transfected cells.Conclusion The EZH2 shRNA vector was successfully constructed,and the stably EZH2-knockdown A2780 cell line was obtained successfully,which provided the foundation for targeting EZH2 in treatment of ovarian cancer.