双sgRNA介导的miR-155基因敲除对FLT3-ITD^(+)AML细胞系的药物敏感性的影响  

Effect of MiR-155 Knockout Mediated by Dual sgRNAs on Drug Sensitivity of FLT3-ITD^(+)AML

作　　者：王凌燕[1] 江佩芳 李佳蒸 胡建达[1] WANG Ling-Yan;JIANG Pei-Fang;LI Jia-Zheng;HU Jian-Da(Department of Hematology Fujian Medical University Union Hospital Fujian Provincial Key Laboratory of Hematology,Fujian Institute of Hematology,Fuzhou 350001,Fujian Province,China)

机构地区：[1]福建医科大学附属协和医院血液科,福建省血液病学重点实验室,福建省血液病研究所,福建福州350001

出　　处：《中国实验血液学杂志》2022年第2期334-340,共7页Journal of Experimental Hematology

基　　金：福建省自然科学基金(2020J05050);国家自然科学基金(81870135,U2005204);福建医科大学启航基金项目(2018QH1036)。

摘　　要：目的:在CRISPR/Cas9基因编辑系统中引入两条不同结合位点的sgRNA共转染FLT3-ITD;AML细胞系MV411获得miR-155基因敲除的MV411细胞,比较单sgRNA转染与双sgRNA转染法进行的miR-155基因敲除的效率及对细胞相关表型的影响。方法:以慢病毒为载体分别构建单转染与双转染的基因敲除细胞,PCR检测基因编辑效率。实时荧光定量PCR法检测miR-155-5p的表达水平。CCK-8法检测细胞增殖能力,并计算细胞对阿霉素与奎扎替尼的药物敏感性。Annexin V-APC/7-AAD双染色检测经阿霉素与奎扎替尼诱导的细胞凋亡水平。结果:CRISPR/Cas9可对miR-155的编码基因进行有效编辑,双sgRNA转染细胞可见明显DNA剪接条带。与单sgRNA转染相比,双sgRNA转染中miR-155-5p的表达水平更低,对阿霉素与奎扎替尼具有更低的IC_(50),更高的药物诱导的细胞凋亡率。结论:双sgRNA转染对miR-155的基因表达的抑制率要高于单sgRNA转染。由双转染基因敲除所引起的细胞抑制增殖、逆转耐药、诱导凋亡等表型更为显著。与单一sgRNA转染相比,双转染sgRNA是一种高效的基因编辑方案。Objective: Two sgRNAs transfected FLT3-ITD+AML cell line MV411 with different binding sites were introduced into CRISPR/cas9 to obtain MV411 cells with mi R-155 gene knockout. To compare the efficiency of mi R-155 gene knockout by single and double sg RNA transfection and their effects on cell phenotypes. Methods: The lentiviral vectors were generated containing either single sg RNA or dual sg RNAs and packaged into lentivirus particles. PCR was conducted to measure gene editing efficiency, and mi R-155 expression was evaluated by q PCR. CCK-8 assay was used to evaluate the cell proliferation, and calculate drug sensitivity of cells to adriamycin and quizartinib. Annexin V-APC/7-AAD staining was used to label cell apoptosis induced by adriamycin and quizartinib. Results: In the dual sg RNAs transfected cells, a cleavage band could be observed, meaning the success of gene editing. Compared with the single sg RNA transfected MV411 cells, the expression level of mature mi R-155-5 p was lower in the dual sg RNA transfected cells. And, dual sg RNA transfected MV411 were more sensitive to adriamycin and quizartinib with lower IC50 and higher apoptosis rate. Conclusion: The inhibition rate of mi R-155 gene expression transfected by dual sg RNA is higher than that by single sg RNA. Dual sg RNA transfection can inhibit cell proliferation, reverse drug resistance, and induce apoptosis more significantly. Compared with single sg RNA transfection, dual sg RNA transfection is a highly efficient gene editing scheme.

关 键 词：MIR-155 CRISPR/Cas9 FLT3-ITD 阿霉素 奎扎替尼 

分 类 号：R733.71[医药卫生—肿瘤]