应用CRISPR/Cas9技术敲除NFE2L2对食管鳞癌KYSE150细胞增殖的影响

Effect of NFE2L2 knockout on proliferation of esophageal cancer KYSE150 cells based on CRISPR/Cas9 technology

目的采用CRISPR/Cas9基因编辑技术构建NFE2L2缺失的食管鳞癌KYSE150(KYSE150 NFE2L2-KO)细胞株,观察NFE2L2敲除对食管鳞癌KYSE150细胞增殖的影响。方法应用CRISPR/Cas9基因编辑技术,构建重组NFE2L2_KO_three_gRNA_Cas9表达载体,将表达载体经转化、扩增、质粒抽提、验证和测序。采用Lipofectamine 2000将重组NFE2L2_KO_three_gRNA_Cas9表达载体转入食管鳞癌KYSE150细胞,经Sanger测序验证转染效率后,培养并筛选单克隆细胞株,获得NFE2L2纯合缺失的食管鳞癌KYSE150单克隆细胞株,并通过Sanger测序、逆转录定量-聚合酶链反应(qRT-PCR)和蛋白印迹法实验验证。将NFE2L2缺失的KYSE150单克隆细胞株设为KYSE150 NFE2L2-KO组,KYSE150单克隆细胞株设为KYSE150组。采用细胞计数试剂法(CCK-8)和克隆形成实验观察NFE2L2敲除对食管鳞癌KYSE150细胞增殖的影响。结果Sanger测序验证结果表明NFE2L2_KO_three_gRNA_Cas9表达载体构建成功以及NFE2L2基因第二外显子上37 kb碱基纯合缺失KYSE150单克隆细胞株成功获得。KYSE150 NFE2L2-KO组,蛋白印迹法未检测到NFE2L2蛋白表达。KYSE150 NFE2L2-KO组中NFE2L2 mRNA表达水平为0.36±0.03,低于KYSE150组的1.04±0.12,t=10.660,P<0.001;CCK-8实验结果显示,48 h时KYSE150 NFE2L2-KO组的细胞增殖活力为4.38±0.31,低于KYSE150组的5.62±0.27,t=4.290,P=0.013;72 h时KYSE150 NFE2L2-KO组的细胞增殖活力为8.77±0.51,低于KYSE150组的12.39±1.50,t=3.231,P=0.032;EdU掺入实验结果显示,KYSE150 NFE2L2-KO组细胞增殖能力为(39.96±2.53)%,低于KYSE150组的(55.60±3.60)%,t=5.019,P=0.007。KYSE150 NFE2L2-KO组细胞克隆形成率为(38.26±2.56)%,低于KYSE150组的(68.93±1.76)%,t=13.950,P<0.001。结论应用CRISPR/Cas9基因编辑技术可成功获得NFE2L2敲除的食管鳞癌KYSE150单克隆细胞系,敲除NFE2L2可抑制KYSE150细胞增殖。

Objective To construct NFE2 L2 knockout KYSE150(KYSE150 NFE2 L2-KO) cell line of esophageal squamous cell carcinoma using CRISPR/Cas9 gene editing technology,and to observe the effect of NFE2 L2 knockout on the proliferation of esophageal squamous cell carcinoma KYSE150 cell line.Methods The recombinant NFE2 L2_ko_three_grna_cas9 expression vector was constructed by CRISPR/Cas9 gene editing technology,and the expression vector was transformed,amplified,plasmid extracted,verified and sequenced.The recombinant NFE2 L2_KO_THREE_GRNA_CAS9 expression vector was transfected into esophageal squamous cell carcinoma KYSE150 cells by using lipofectamine 2000.After the transfection efficiency was verified by the Sanger sequencing,the monoclonal cell line KYSE150 with homozygous deletion of NFE2 L2 was obtained by culture and screening.The Sanger sequencing,quantitative reverse transcription-polymerase chain reaction(qRT-PCR)and western blot were used to verify the obtained monoclonal cell line KYSE150 of esophageal squamous cell carcinoma with homozygous deletion of NFE2 L2.The KYSE150 monoclonal cell line with NFE2 L2 deletion was set as KYSE150 NFE2 L2-KO group,and the KYSE150 monoclonal cell line was set as KYSE150 group.Cell counting kit-8(CCK-8),EdU incorporation assays and clone formation assay were used to observe the effect of NFE2 L2 knockout on proliferation of KYSE150 cells in esophageal squamous cell carcinoma.Results Comparative analyses of Sanger sequencing results confirmed the successful construction of NFE2 L2_KO_three_gRNA_Cas9 expression vector and the successful establishment of KYSE150 monoclonal cell line with 37 kb base homozygous deletion on Exon2 of NFE2 L2 gene.In KYSE150 NFE2 L2-KO group,western blot could not detect protein expression of NFE2 L2.The mRNA expression level of NFE2 L2 in KYSE150 NFE2 L2-KO group was 0.36±0.03,which was significantly lower than that in KYSE150 group of 1.04±0.12,with statistical significance(t=10.660,P<0.001).CCK-8 detected the cell proliferation activity,the cell proliferation activity of KYSE150 NFE2 L2-KO group at 48 hwas 4.38±0.31,which was lower than 5.62±0.27 of KYSE150 group,t=4.290,P=0.013.At 72 h,the cell proliferation activity of KYSE150 NFE2 L2-KO group(8.77±0.51)was significantly lower than that of KYSE150 group(12.39±1.50),t=3.231,P=0.032.The results of EdU incorporation assays showed that the cell proliferation ability of KYSE150 NFE2 L2-KO group was(39.96±2.53)%,lower than that of KYSE150 group(55.60±3.60)%,t=5.019,P=0.007.The rate of cell clone formation in KYSE150 NFE2 L2-KO group(38.26±2.56)% was significantly lower than that in KYSE150 group(68.93±1.76)%,t=13.950,P<0.001.Conclusion Using CRISPR/Cas9 gene editing technique,NFE2 L2 knockout KYSE150 monoclonal cell line of esophageal squamous cell carcinoma is successfully obtained,and NFE2 L2 knockout significantly inhibite the proliferation of KYSE150 cells.