siRNA沉默EZH2对HL-60细胞迁移、增殖能力的影响及其机制  

Effect and mechanism of siRNA silencing EZH2 on migration and proliferation ability of HL-60 cells

作　　者：朱秋花 潘学谊[1] 曾文彬 周兰兰[1] 关则兵 ZHU Qiuhua;PAN Xueyi;ZENG Wenbin;ZHOU Lanlan;GUAN Zebing(Department of Hematology,First Affiliated Hospital of Guangdong Pharmaceutical University,Guangzhou,Guangdong 510080,China)

机构地区：[1]广东药科大学附属第一医院血液内科,广州510080

出　　处：《重庆医学》2021年第10期1653-1657,1662,共6页Chongqing medicine

基　　金：广东省自然科学基金项目(2017A030313664);广州市越秀区科技计划项目(2017-W S-008)。

摘　　要：目的探讨人类同源基因2(EZH2)对人急性早幼粒细胞白血病细胞系HL-60细胞迁移、增殖能力的影响及其机制。方法通过慢病毒转染技术用siRNAs沉默HL-60细胞的EZH2基因,用实时荧光定量PCR(qRT-PCR)及Western blot验证3个不同序列的siRNA敲除EZH2的效果并筛选出最佳序列。随后用CCK-8和流式细胞术分别检测细胞增殖活性和凋亡,并用Transwell迁移小室试验检测细胞的迁移能力,用Western blot方法检测细胞迁移、增殖及凋亡相关蛋白的表达水平。结果在HL-60细胞中用siRNAs敲除EZH2后其增殖能力下降(P<0.05),凋亡增加(P<0.05)。Transwell迁移小室试验结果显示,HL-60细胞敲除EZH2组(siEZH2组)的下室细胞比例比HL-60野生株细胞组(wild组)和空病毒组(NC组)明显降低;苏木素染色后发现siEZH2组膜上细胞基数明显低于wild组和NC组。Western blot检测显示,siRNA沉默EZH2后细胞迁移相关蛋白基质金属蛋白酶2(MMP-2)表达下调,E-钙黏蛋白(E-cadherin)上调,增殖及凋亡相关蛋白B淋巴细胞瘤-2(Bcl-2)基因下调,而半胱氨酸天冬氨酸蛋白酶3(caspase-3)上调。结论在HL-60细胞中敲除EZH2基因后其增殖、迁移能力下降,凋亡增加。Objective To investigate the effect and mechanism of EZH2 on the migration and proliferation ability of acute promyelocytic leukemia HL-60 cells.Methods siRNAs were used to silence the EZH2 gene in HL-60 cells by lentiviral transfection.The effect of three different sequences of siRNA knocking out EZH2 was confirmed by quantitative reverse transcriptase-polymerase chain reaction(QRT-PCR)and Western blot,moreover the optimal sequence was screened out.Subsequently,the cell proliferation activity and apoptosis were detected by cell counting kit-8(CCK-8)and flow cytometry,the cell migration ability was detected by Transwell migration assay,and the expression levels of migration,proliferation and apoptosis associated proteins were detected by Western blot.Results After siRNAs knocking out EZH2 in HL-60 cells,the proliferation ability was decreased(P<0.05)and apoptosis was increased(P<0.05).The Transwell migration chamber test results showed that the ratio of migrated cells in the lower compartments in EZH2 group(siEZH2 group)was significantly decreased compared with that in the HL-60 wild cells group(wild group)and empty virus group(NC group),and the cells base number on the membrane in the siEZH2 group was significantly lower than that in the wild group and NC group after Hematoxylin staining.The Western blot detection found that after silencing EZH2 by siRNA,the expression of migration-related protein MMP-2 was down-regulated and E-cadherin was up-regulated;and the proliferation and apoptosis related protein B-cell lymphoma-2(Bcl-2)gene was down-regulated,while caspase-3 was up-regulated.Conclusion After EZH2 knocking out EZH3 gene in HL-60 cells,its proliferation and migration ability is decreased and apoptosis is increased.

关 键 词：急性早幼粒细胞白血病 HL-60 人类同源基因2 细胞迁移 髓外浸润 

分 类 号：R557+.3[医药卫生—血液循环系统疾病]