利用CRISPR/Cas9系统对人A549肺癌细胞<em>NRF</em>2基因的稳定敲除及其功能研究

龚美玲; 张琳琳; 郑翠侠

doi:10.19401/j.cnki.1007-3639.2019.11.003

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利用CRISPR/Cas9系统对人A549肺癌细胞NRF2基因的稳定敲除及其功能研究

更新时间：2026-01-27

- 利用CRISPR/Cas9系统对人A549肺癌细胞NRF2基因的稳定敲除及其功能研究
- Stable knockout ofNRF2 gene in human A549 lung cancer cells by CRISPR/Cas9 system and its functional research
- 中国癌症杂志 2019年第11期页码：855-861
- 作者机构：
  
  同济大学附属杨浦医院呼吸内科,上海,200090
- 作者简介：
- 基金信息：
  
  国家自然科学基金（8177100416）。
- DOI：10.19401/j.cnki.1007-3639.2019.11.003
  中图分类号：
- 网络出版：2019-12-09，
  
  纸质出版：2019-12-09
- 稿件说明：
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龚美玲, 张琳琳, 郑翠侠. 利用CRISPR/Cas9系统对人A549肺癌细胞

GONG Meiling, ZHANG Linlin, ZHENG Cuixia. Stable knockout ofNRF2 gene in human A549 lung cancer cells by CRISPR/Cas9 system and its functional research[J]. China Oncology, 2019, (11): 855-861.
龚美玲, 张琳琳, 郑翠侠. 利用CRISPR/Cas9系统对人A549肺癌细胞 DOI： 10.19401/j.cnki.1007-3639.2019.11.003.

GONG Meiling, ZHANG Linlin, ZHENG Cuixia. Stable knockout ofNRF2 gene in human A549 lung cancer cells by CRISPR/Cas9 system and its functional research[J]. China Oncology, 2019, (11): 855-861. DOI： 10.19401/j.cnki.1007-3639.2019.11.003.

摘要

背景与目的：CNC-bZIP是核转录因子E2相关因子2［nuclear factor（erythroid-derived 2）-like 2，NRF2］近羧基端一个亮氨酸拉链结构，该区域是DNA结合以及NRF2与小分子肌腱纤维肉瘤（small masculoaponeurotic fibrosarcoma，sMaf）蛋白结合形成二聚体所必需的区域，随后该区域与抗氧化反应元件（antioxidant response element，ARE）结合，激活NRF2下游靶基因的转录表达，从而增加细胞抗氧化应激能力。构建CRISPR/Cas9慢病毒系统，获得了A549细胞中NRF2基因稳定敲除株并进行了功能研究。方法：针对该结构上下游设计一对sgRNA，与pLenti CRISPR v2载体连接，通过包装慢病毒的方式构建A549稳定敲除细胞系。根据测序图谱及蛋白质印迹法（Western blot）验证敲除效果。然后通过实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）、Western blot检测、克隆形成实验、细胞计数试剂盒（cell counting kit-8，CCK-8）、划痕实验、Transwell实验比较A549细胞株NRF2敲除与不敲除的功能表达。结果：显示A549肺癌细胞中NRF2基因稳定敲除株构建成功；NRF2敲除株下游靶基因mRNA表达及蛋白水平明显减少，且能明显降低A549肺癌细胞的增殖、迁移、侵袭能力。结论：利用CRISPR/Cas9慢病毒系统获得了高效永久NRF2基因敲除的肺癌细胞系，该基因敲除后A549肺癌细胞的增殖、迁移、侵袭能力都显著降低。

Abstract

Background and purpose: CNC-bZIP is a leucine zipper structure near the carboxyl terminal of homo sapiens nuclear factor (erythroid-derived 2)-like 2 (NRF2)

which is necessary for DNA binding

and associated with NRF2 dimerization partners and the small masculoaponeurotic fibrosarcoma (sMaf) proteins. Then this structure binds to the antioxidant response element (ARE) for activating the transcriptional expression of a downstream target gene of NRF2

thereby increasing the cell’s ability to resist oxidative stress. This study aimed to establish the CRISPR/Cas9 lentiviral system

and produce stable NRF2 gene knockout A549 cell line. Methods: A pair of sgRNAs were designed for upstream and downstream structures

and ligated with the pLenti CRISPR v2 vector to construct the stable knockout A549 cell line. The effect was verified by sequencing maps and Western blot. The functions of NRF2 knockout cell line and the A549 cell line without gene knockout were compared by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR)

Western blot

colony formation experiments

cell counting kit-8 (CCK-8)

wound-healing assay and transwell assay. Results: As expected

the results showed that the stable A549 cell line with deficient NRF2 expression was successfully obtained. The mRNA expression and protein level of the downstream target genes of NRF2 knockout strains were significantly reduced

and the proliferative

migration and invasion abilities were greatly decreased. Conclusion: CRISPR/Cas9 lentiviral system was used to obtain a highly efficient permanent NRF2 knockout lung cancer cell line

and the proliferation

migration and invasion abilities of A549 lung cancer cells were greatly reduced after NRF2 gene knockout.

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利用CRISPR/Cas9系统对人A549肺癌细胞NRF2基因的稳定敲除及其功能研究

Stable knockout ofNRF2 gene in human A549 lung cancer cells by CRISPR/Cas9 system and its functional research

DOI：10.19401/j.cnki.1007-3639.2019.11.003

摘要

Abstract

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