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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 small hairpin RNA (shRNA) recombinant expression vector targeting zeste gene enhancer homolog 2 (EZH2) and to explore the role of EZH2 gene in colorectal cancer cells. Methods Two DNA sequences with short hairpin structure were designed according to the sequence of EZH2cDNA. Recombinant vector was constructed with vector pGFP-V-RS and transfected into SW480 cells. They were randomly divided into negative control group and gene silencing group. The inhibitory effect was detected by RTPCR and Western blotting. Cell proliferation was detected by MTT assay. Results Interfering plasmids that inhibited EZH2 gene expression were successfully constructed. The expression of EZH2 mRNA in the negative control group was 5.8-fold more than that in the gene silencing group (P <0.01). The protein expression of EZH2 in the gene silencing group was significantly lower than that of the negative control group (P <0.05). Compared with the negative control group, the cell growth of gene silencing group was significantly inhibited (P <0.05). Conclusion EZH2 target shRNA recombinant expression vector was successfully constructed and could significantly inhibit the expression of EZH2 gene, which provided a basis for further study of the mechanism of EZH2 gene in the tumor.