用CRISPR/Cas9技术编辑KLF4基因对GES-1细胞生物学行为的影响

Effect of KLF4 gene edited by CRISPR/Cas9 technique on the biological behavior of human gastric epithelial GES-1 cells

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 plasmid for knockout of Kruppel-like factor 4 （KLF4） gene by using CRISPR/ Cas9 gene editing method and examine its impact on biological behavior of human GES-1 cells. Methods Synthe- sized KLF4 gene targeting sgRNA oligos were inserted into CRISPR/Cas9 plasmid vector of pX459. After transfor- mation, clone isolation, and amplification, DNA sequencing confirmed plasmid DNA, termed pX459-KLF4-sgRNA,was used to transfect human gastric epithelial GES-1 cells in vitro. After transfection, GES-1 cells were first select- ed in cell-cultured medium containing puromycin, and then the selected GES-1 cells were replated into 100 mm di- shes for clonal cell culture. Protein samples extracted from individual cell clones were used for Western blot analy- sis of KLF4 expression. Biological assays of plate clone formation, MTr cell proliferation, and transwell cell migra- tion were used to compare the cellular behavior changes among GES-1 ceils of parental, control clone or clone with KLF4 gene knockdown. Results DNA sequence analysis confirmed that KLF4 targeting sgRNA sequences were successfully constructed into of pX459 vector. Transfection of the resultant vector of pX459-KLF4-sgRNA into GES- 1 cells led to KLF4 gene knockdown indicated by drastic reduction of KLF4 protein expression, significant biologi- cal behavior changes including increased cell proliferation, clone formation, and migration abilities. Conclusion We successfully constructed human KLF4 gene editing plasmid vector based on CRISPR/Cas9 technique, and the biological behavior changes of the established GES-1 cell line with drastic KLF4 knockdown confirmed the tumor suppressive function of KLF4.