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目的采用CRISPR/Cas9基因编辑技术,构建靶向KLF4的基因编辑质粒,建立CRISPR/Cas9-sg KLF4人正常胃黏膜GES-1细胞株,观察KLF4基因敲除效果及其对细胞生物学行为的影响。方法设计靶向KLF4基因的sgRNA序列,将合成的片段插入到CRISPR/Cas9质粒骨架载体p X459中,再将重组后的质粒做转化,挑取克隆,扩增后提质粒进行测序验证正确性。将构建好的重组质粒体外转染入人胃黏膜细胞GES-1中,利用嘌呤霉素进行筛选获得KLF4敲除的克隆细胞,应用Western blot技术检测未转染细胞和克隆细胞中KLF4蛋白的表达情况,平板克隆实验检测KLF4敲低后克隆形成能力,MTT实验检测其增殖能力,Transwell实验检测其迁移能力。结果经测序验证,成功构建了靶向KLF4的重组质粒p X459-KLF4-sgRNA,经Western blot方法验证,转染该重组质粒后人胃黏膜上皮细胞株GES-1的KLF4蛋白表达量明显降低,行为学实验结果表明,GES-1细胞敲低KLF4基因后克隆形成能力、增殖能力以及迁移能力都明显增强。结论成功构建了靶向KLF4的CRISPR/Cas9基因编辑质粒载体及KLF4基因敲低的稳定细胞株GES-1,KLF4基因敲低后GES-1细胞生物学行为的改变证实其抑癌基因活性。
Objective To construct a gene editing plasmid targeting KLF4 by CRISPR / Cas9 gene editing technology and establish a human gastric cancer cell line GES-1 with CRISPR / Cas9-sg KLF4 gene knockout effect and its effect on cell biological behavior . Methods The sgRNA sequence targeting KLF4 gene was designed and inserted into the CRISPR / Cas9 plasmid vector pX459. The recombinant plasmids were transformed and the clones were picked. After amplification, the plasmid was sequenced to verify the correctness. The constructed recombinant plasmids were transfected into human gastric mucosal cells GES-1 in vitro and screened with puromycin to obtain KLF4 knockout cloned cells. The expression of KLF4 protein in untransfected and cloned cells was detected by Western blot The clonal formation of KLF4 knockdown was detected by plate clone assay. The proliferation of KLF4 knockdown was detected by MTT assay. The transwell assay was used to detect the migration ability. Results The recombinant plasmid pX459-KLF4-sgRNA targeting KLF4 was successfully constructed and verified by sequencing. The expression of KLF4 protein in human gastric mucosal epithelial cell line GES-1 was significantly decreased after transfected with the recombinant plasmid. The results of behavioral experiments showed that the ability of KLF4 gene knockdown in GES-1 cells after clonogenicity, proliferation and migration were significantly enhanced. Conclusion The recombinant plasmid of CRISPR / Cas9 targeting KLF4 and GES-1, a stable cell line with knockdown of KLF4 gene, were successfully constructed. The biological behavior of GES-1 cells knocked down by KLF4 gene was confirmed to be tumor suppressor gene.