利用CRISPR/Cas9基因编辑技术构建敲除4EBP1基因的乳腺癌细胞系  

Construction of 4EBP1 knockout breast cancer cell lines using CRISPR/Cas9 gene editing technology

作　　者：鄱宛玉 张哲 罗国兰 黄芳 郑红[1] 李山虎 Yan Wanyu;Zhang Zhe;Nuo Guolan;Huang Fang;Zheng Hong;Li Shanhu(School of Basic Medicine,Anhui Medical University,Hefei 230032,China)

机构地区：[1]安徽医科大学基础医学院,合肥230032

出　　处：《中华乳腺病杂志（电子版）》2023年第3期143-150,共8页Chinese Journal of Breast Disease(Electronic Edition)

基　　金：安徽省高校协同创新项目(GXXT-2021-063)。

摘　　要：目的利用规律性重复短回文序列簇/相关基因9(CRISPR/Cas9)编辑技术构建真核翻译起始因子4E结合蛋白1(4EBP1)基因敲除的人乳腺癌MCF-7、ZR-75-1细胞系。方法设计能特异性针对4EBP1基因的特异性向导RNA(sgRNA),以lentiCRISPRv2质粒为骨架构建能表达sgRNA和Cas9蛋白的重组质粒。测序鉴定后将重组质粒与逆转录病毒包装质粒pMD2.G、psPAX2共同转入HEK293T细胞进行慢病毒包装,收集病毒上清感染人乳腺癌MCF-7、ZR-75-1细胞。利用嘌呤霉素筛选4EBP1表达缺失的MCF-7、ZR-75-1细胞,DNA测序和Westernblot验证。在MCF-7、ZR-75-1野生型与4EBP1基因敲除的细胞中转人pcDNA3-rluc-poli-IRESfluc质粒,并用双荧光素酶报告基因实验检测4EBP1、4EBP1不同突变体对帽子依赖翻译的影响,以海肾荧光素酶与萤火虫荧光素酶比值(R/F比值)来表示帽子依赖的翻译水平。R/F比值的组间比较采用t检验,两两比较采用LSD法。结果Western blot结果显示构建的3种lentiCRISPRv2-4EBP1-KO重组质粒转染MCF-7、ZR-75-1细胞后,4EBP1蛋白的表达水平均明显降低,说明4EBP1基因敲除成功。DNA测序发现MCF-7细胞的DNA序列中sgRNA出现了T碱基的插入突变,在ZR-75-1细胞DNA序列中sgRNA出现了GC碱基的缺失突变,证明细胞基因组DNA中的基因编辑成功。野生型和4EBP1基因敲除的ZR-75-1细胞中测得的R/F比值分别为1.83±0.10和5.48±0.33,组间比较差异有统计学意义(t=-18.338,P<0.001)。野生型和4EBP1基因敲除的MCF-7细胞中测得的R/F比值分别为1.17±0.06和2.03±0.20,组间比较差异有统计学意义(t=-7.167,P<0.001)。4EBP1基因敲除、敲除后分别转染4EBP1、plv-neo-4EBP1-37/46和plv-neo-4EBP1-4A的4种ZR-75-1细胞中测得的R/F比值分别为5.48±0.33、3.83±0.10、3.53±0.23和1.87±0.05,组间比较差异有统计学意义(F=151.995,P<0.001);4种MCF-7细胞中测得的R/F比值分别为2.03±0.200、1.37±0.14、1.90±0.19和1.20±0.17组间比较差异有统计学意Objective To construct the eukaryotic translation initiation factor 4E binding protein 1(4EBP1)gene knockout human breast cancer MCF-7 and ZR-75-1 cell lines using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated 9(Cas9)gene editing technology.Methods We designed a sequence-specific guide RNA(sgRNA)that specifically targeted the 4EBP1 gene,and used the lentiCRISPR v2 plasmid as a backbone to construct a recombinant plasmid that expressed sgRNA and Cas9 protein.After sequencing and identification,the recombinant plasmid was co-transferred into HEK 293T cells with retroviral packaging plasmids pMD2.G and psPAX2 for lentiviral packaging,and viral supernatants at 24 h and 48 h were collected to infect human breast cancer MCF-7 and ZR-75-1 cells.MCF-7 and ZR-75-1 cells with deletion of 4EBP1 were screened out using puromycin,and verified by DNA sequencing and Western blot analysis.The MCF-7,ZR-75-1 wild-type and 4EBP1 knockout cells were transfected with pcDNA3-rluc-poli-IRESfluc plasmid.The effects of different mutants of 4EBP1 on cap-dependent translation were detected using a dual luciferase reporter gene assay and the cap-dependent translation level was expressed as the ratio of renilla luciferase to firefly luciferase(R/F ratio).The t-test was used to compare the R/F ratio between groups,and the LSD method was used for pairwise comparisons.Results Western blot analysis showed that the expression levels of 4EBP1 protein were significantly reduced in MCF-7 and ZR-75-1 cells transfected with the three constructed lentiCRISPR v2-4EBP1-KO plasmids,indicating successful knockdown of 4EBP1 gene.DNA sequencing revealed a T-base insertion of sgRNA in the DNA sequence of MCF-7 cells,and a GC-base deletion of sgRNA in the DNA sequence of ZR-75-1 cells,demonstrating the success of gene editing in the cellular genomic DNA.The R/F ratios measured in wild-type and 4EBP1 knockout ZR-75-1 cells were 1.83±0.10 and 5.48±0.33,respectively,suggesting a significant difference between two groups(t=-

关 键 词：真核翻译起始因子4E结合蛋白1 人类 乳腺肿瘤 细胞系 肿瘤 

分 类 号：R737.9[医药卫生—肿瘤]