应用CRISPR/Cas9技术建立敲除AIFM1基因的稳定肺癌细胞株  

Application of CRISPR/Cas9 technology to establish a stable lung cancer cell line with knockdown of AIFM1 gene

作　　者：汤喜连 李宁 余华军 陈小谊 黄小琴 贾玉芳 涂名进 何柳燕 伍俊 张海涛 TANG Xilian;LI Ning;YU Huajun;CHEN Xiaoyi;HUANG Xiaoqin;JIA Yufang;TU Mingjin;HE Liuyan;WU Jun;ZHANG Haitao(Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics,School of Medical Technology,Guangdong Medical University,Dongguan,Guangdong 523000,China;Department of Respiratory Medicine,Affiliated Hospital of Guangdong Medical University,Zhanjiang,Guangdong 524023,China;Department of Biochemistry and Molecular Biology,Guangdong Medical University,Zhanjiang,Guangdong 524023,China;Guangdong Medical University Laboratory Animal Center,Zhanjiang,Guangdong 524023,China;Peptide and Protein Research and Application Key Laboratory of Guangdong Medical University,Zhanjiang,Guangdong 524023,China)

机构地区：[1]广东医科大学医学技术学院广东省医学分子诊断学重点实验室,广东东莞523000 [2]广东医科大学多肽与蛋白质研究应用重点实验室,广东湛江524023 [3]广东医科大学生物化学与分子生物学教研室,广东湛江524023 [4]广东医科大学实验动物中心,广东湛江524023 [5]广东医科大学附属医院呼吸疾病研究所,广东湛江524023

出　　处：《热带医学杂志》2023年第10期1346-1350,1363,1336,共7页Journal of Tropical Medicine

基　　金：广东省自然科学基金(2021A1515011621);广东医科大学学科建设项目(4SG21012G)

摘　　要：目的利用CRISPR/Cas9技术在肺癌细胞系A549和NCI-H3255中敲除凋亡诱导因子1(AIFM1)基因,并通过筛选,获得稳定的细胞株。方法利用在线数据库GEPIA、CPTAC、Prognosis和SangerBox分析AIF在肺癌的表达和功能。以AIFM1基因为靶点,设计向导RNA(sgRNA)并利用CRISPR RGEN Tools分析其脱靶效率,CRISPR/Cas9技术敲除肺癌细胞的AIFM1基因和倍比稀释法筛选稳定的单克隆细胞株。采用Western blot检测细胞AIF表达水平。结果TCGA和GTEx数据集结果显示,AIF蛋白在肺癌组织阳性表达率比正常组织高,差异有统计学意义(P<0.05);单因素cox和多因素cox分析发现,pT分期和pN分期是独立危险因素(P<0.05);CPTAC数据库显示,与正常阶段相比,AIF蛋白在肺癌的不同分级分期阶段的表达增加(P<0.05)。AIFM1基因在TCA循环、亨廷顿氏症疾病、氨基酸-核苷酸-糖的代谢和氧化性磷酸化通路中富集显著,差异有统计学意义(|NES|>1,P<0.05);3条sgRNA的序列脱靶效率均为0;与野生型肺癌细胞株相比,用CRISPR/Cas9基因编辑技术修饰的肺癌细胞未检测到表达AIF蛋白,且筛选的杀死A549和NCI-H3255的嘌呤霉素浓度分别为0.60~0.80μg/mL和0.60μg/mL。结论AIF在肺癌中表达上调。利用CRISPR/Cas9技术成功建立AIFM1基因敲除的肺癌细胞株,这将为研究AIF在肺癌发生发展机制提供合适的研究材料。Objective To knock down the apoptosis inducing factor 1(AIFM1)gene in lung cancer cell lines A549 and NCI-H3255 using CRISPR/Cas9 technology,and obtain stable cell lines by screening.Methods The expression and function of AIF in lung cancer were analyzed using the online databases GEPIA,CPTAC,Prognosis and SangerBox.AIFM1 gene was used as the target,guide RNA(sgRNA)was designed and analyzed for off-target efficiency using CRISPR RGEN Tools,AIFM1 gene was knocked down from lung cancer cells by CRISPR/Cas9 technology and stable monoclonal cell lines were screened by fold dilution method.The cellular AIF expression level was detected by western blot.Results The TCGA and GTEx datasets showed that the expression of AIF protein in lung cancer tissues was higher than that in normal tissues,the difference was statistically significant(P<0.05).Univariate Cox and multivariate Cox analyses revealed that pT stage and pN stage were independent risk factors(P<0.05).The CPTAC database showed that the expression of AIF proteins was increased in different graded staging stages of lung cancer compared to normal stages(P<0.05).The AIFM1 gene was significantly enriched in the TCA cycle,Huntington's disease,amino acid-nucleotide-sugar metabolism,and oxidative phosphorylation pathways,with statistical significance(|NES|>1,P<0.05).The off-target efficiency of the three sgRNA sequences was all 0.Compared to the wild-type lung cancer cell line,the lung cancer cells modified by CRISPR/Cas9 gene editing technology did not express AIF protein,and the selected concentrations of mercaptopurine for killing A549 and NCI-H3255 were 0.60-0.80μg/mL and 0.60μg/mL,respectively.Conclusions AIF was upregulated in lung cancer expression.AIFM1 knockdown lung cancer cell line was successfully established using CRISPR/Cas9 technology.This study provided suitable research material for studying the mechanism of AIF in lung carcinogenesis and development.

关 键 词：CRISPR/Cas9 AIFM1基因 基因敲除 肺癌 

分 类 号：R734.2[医药卫生—肿瘤]