利用CRISPR-Cas9基因编辑技术敲除HOXA5基因对急性髓系白血病细胞增殖的影响
Effect of Knocking Out HOXA5 Gene by CRISPR-Cas9-Mediated Gene Editing Technique on Proliferation of Acute Myeloid Leukemia Cells
满建呈 1成娟 1赵丽1
作者信息
- 1. 兰州大学第一临床医学院,兰州大学第一医院中心实验室,甘肃省血液病遗传研究重点实验室,甘肃兰州 730000
- 折叠
摘要
目的:通过CRISPR-Cas9基因编辑技术构建稳定敲除HOX45基因的急性髓系白血病(AML)细胞株,明确HOXA5基因敲除对AML细胞增殖的影响,初步探索HOXA5基因在AML发病中的作用.方法:通过荧光定量PCR(qRT-PCR)和Western blot技术分别检测血液系统非肿瘤患者与初诊AML患者骨髓单个核细胞(BMMC)中HOXA5的表达;应用CRISPR-Cas9基因编辑技术构建稳定敲除HOXA5基因的AML细胞株KO-HOXA5-THP-1,通过qRT-PCR和Western blot检测KO-HOXA5-THP-1细胞中HOXA5的表达,并采用CCK-8实验检测细胞增殖情况.结果:与血液系统非肿瘤患者相比,初诊AML患者骨髓单个核细胞中HOXA5基因及蛋白水平均显著升高(P＜0.05).应用CRISPR-Cas9基因编辑技术能够获得稳定敲除HOXA5基因的细胞株,而且敲除HOXA5基因后的THP-1细胞株的增殖能力显著下降(P＜0.05).结论:HOXA5在AML细胞中高表达,敲除HOXA5能够显著影响AML细胞的增殖能力,这为急性髓系白血病的精准治疗提供了一个新的潜在治疗靶点.
Abstract
Objective:To construct a acute myeloid leukemia(AML)cell line in which HOXA5 gene is stably knocked out by CRISPR-Cas9-mediated gene editing technique,so as to clarify the effect of HOXA5 gene knockout on the proliferation of AML cells,and preliminarily explore the role of HOXA5 gene in the pathogenesis of AML.Methods:The expression of HOXA5 in bone marrow mononuclear cells(BMMC)of non-tumor hematological patients and newly diagnosed AML patients was detected by quantitative real-time PCR(qRT-PCR)and Western blot,respectively.The AML cell line KO-HOXA5-THP-1 was constructed in which HOXA5 gene was knocked out by CRISPR-Cas9-Mediated gene editing technique,and the knockout of HOXA5 gene was verified by qRT-PCR and Western blot,and the cell proliferation was detected by CCK-8 assay.Results:Compared with non-tumor hematological patients,the levels of HOXA5 gene and protein in BMMC of newly diagnosed AML patients were significantly increased(P＜0.05).The stable HOXA5 knockout cell line can be obtained by CRISPR-Cas9-Mediated gene editing technique,and the proliferation ability of THP-1 cells with HOXA5 gene knockout was significantly decreased(P＜0.05).Conclusion:HOXA5 is highly expressed in AML cells,and knocking out HOXA5 can significantly affect the proliferation ability of AML cells,which provides a new potential therapeutic target for the precise treatment of AML.
关键词
急性髓系白血病/HOXA5基因/CRISPR-Cas9Key words
acute myeloid leukemia/HOXA5 gene/CRISPR-Cas9引用本文复制引用
基金项目
甘肃省自然科学基金(21JR1RA114)
兰州大学第一医院院内基金学科交叉研究基金(ldyyyn2020-89)
出版年
2024
NETL
NSTL
维普
万方数据