摘要
目的:运用CRISPR/Cas9基因编辑技术,建立miR-362基因敲除的95-D肺癌细胞株,并研究miR-362在肿瘤中的调控作用。方法:针对人源miR-362基因序列设计gRNA,构建px330-gRNA载体;T7E1 assay确定gRNA的有效性。分别扩增miR-362上下游同源臂序列构建donor载体。利用脂质体将CRISPR系统和donor载体共转至人肺癌细胞系95-D,通过同源重组方法将筛选标志基因整合至基因组中,通过流式分选以及q PCR方法检测筛选出的细胞中miR-362表达水平。利用Transwell分析miR-362对细胞运动能力的影响。结果:与95-D细胞相比,95-D-Knock Down细胞中miR-362表达水平显著降低,且迁移侵袭能力分别下降了53.1%(P=0.000 6)和48.3%(P=0.000 2)。结论:利用CRISPR/Cas9系统成功构建了miR-362基因敲除的95-D细胞,miR-362可促进细胞的运动能力,为后续研究miR-362在肿瘤中的作用机制和功能奠定了基础。
Objective： to construct miR-362 knockdown 95-D cells by using CRISPR/Cas9 genome engineering technology, and study the function of miR-362 in cancer. Methods： gRNA sequences targeting the miR-362 gene were selected. Px330-gRNA recombination plasmids were constructed and the validity were evaluated by T7E1 assay. Left and right arms of miR-362 were amplified from genomic DNA by PCR, and sequentially cloned into the donor vector. The CRISPR/Cas9 system and donor vector were co-transfection into 95-D cells, and the selection system that allows for marker genes were integrated into the genome through homologous recombination （HR）. The expression of miR-362 of 95-D-KnockDown cells sorted through FACS was detected by qPCR, and the migration and invasion were determined by Transwell assay. Results：Compared with 95-D cells, the expression of miR-362 in 95-D-KD cells was significantly down-regulated, and down-regulation of miR-362 expression can suppress the cell migration and invasion capacity of 95-D-KD cells. Conclusion： The miR-362 knockdown lung cancer cell line（95-D-KD cells）had been successfully constructed by using CRISPR/ Cas9 system, which lays the foundation for further study of the mechanism and function of miR-362 in cancers.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2017年第11期94-100,共7页
China Biotechnology
基金
国家自然科学基金资助项目(81572817)
