基于生物信息学的DNA甲基转移酶1相关差异基因筛选及其对AML细胞增殖、凋亡、侵袭的影响  

Bioinformatics-based screening DNA methyltransferase 1-related differential genes and their effects on AML cell proliferation,apoptosis,and invasion

作　　者：余静静[1] 郭沙 岳晶晶[2] 黄彩娟[2] 曲建华[1] YU Jingjing;GUO Sha;YUE Jingjing;HUANG Caijuan;QU Jianhua(Hematology Center,the First Affiliated Hospital of Xinjiang Medical University,Urumqi 830000,Xinjiang,China;Department of Hematology,the First Affiliated Hospital of Henan University of Science and Technology,Luoyang 471003,Henan,China)

机构地区：[1]新疆医科大学第一附属医院血液病中心,新疆乌鲁木齐830000 [2]河南科技大学第一附属医院血液科,河南洛阳471003

出　　处：《贵州医科大学学报》2024年第12期1739-1748,共10页Journal of Guizhou Medical University

基　　金：国家自然科学基金(82160034);自治区“天山创新团队计划”项目(2022D14008);“天山英才”医药卫生高层次人才培养计划项目(TSYC202301A006)。

摘　　要：目的筛选新的DNA甲基转移酶1(DNMT1)相关基因并探讨其对AML细胞增殖、凋亡、侵袭的影响。方法在癌症基因组图谱(TCGA)数据库中检索AML患者数据,采用生物信息学分析方法筛选与DNMT1相关的差异表达激酶;采用小干扰RNA(siRNA)干扰技术干扰筛选出的细胞外调节蛋白激酶(ERK1)、同源结构域相互作用蛋白激酶2(HIPK2)及糖原合酶激酶3β(GSK3β)表达,得si-ERK1、si-HIPK2及si-GSK3β细胞;取培养至对数生长期的AML细胞HL-60分为control组(无处理)、si-NC组(空白质粒转染)、si-ERK1组、si-HIPK2组及si-GSK3β组,采用细胞计数试剂盒(CCK-8)和流式细胞术分别检测细胞活力和凋亡率,采用实时荧光定量核酸扩增检测(RT-qPCR)和Western blot检测HIPK2、GSK3β、ERK1、信号转导及转录激活蛋白3(STAT3)、磷酸化STAT3(P-STAT3)、雅努斯激酶(JAK)、磷酸化JAK(P-JAK)、磷酸肌醇3-激酶(PI3K)、磷酸化PI3K(P-PI3K)、丝氨酸/苏氨酸激酶Akt(AKT)、磷酸化AKT(P-AKT)信使RNA(mRNA)及蛋白的表达。结果生物信息学分析结果显示,低DNMT1与高DNMT1组AML患者细胞周期依赖性激酶1(CDK1)、酪蛋白激酶2α1(CSNK2A1)、丝裂原活化蛋白激酶1(MAPK1)、MAPK14、CDK4、ERK1、HIPK2及GSK3β水平比较,差异有统计学意义(P<0.05),最终选择ERK1、HIPK2及GSK3β进行后续研究;与control组和si-NC组比较,干扰HL-60细胞中HIPK2、GSK3β及ERK1蛋白表达下降(P<0.001);与control组和si-NC组比较,干扰HL-60细胞中ERK1、HIPK2或GSK3β的表达后细胞活力下降(P<0.05),细胞凋亡率上升(P<0.05);RT-qPCR结果显示,与control组和si-NC组比较,干扰HL-60细胞中ERK1、HIPK2或GSK3β表达后STAT3、JAK、PI3K及AKT mRNA表达下降(P<0.05);Western blot结果显示,与control组和si-NC组比较,干扰HL-60细胞中ERK1、HIPK2或GSK3β表达后P-STAT3、P-JAK、P-PI3K及P-AKT蛋白水平降低(P<0.05)。结论筛选出新的DNMT1相关基因ERK1、HIPK2及GSK3β,其表达可抑制AML细胞增殖和侵袭、并�Objective To screen novel DNA methyltransferase 1(DNMT1)-related genes and explore their effects on cell proliferation,apoptosis and invasion of acute myeloid leukemia(AML).Methods AML patient data was retrieved from the Cancer Genome Atlas(TCGA)database.Bioinformatics analysis was used to screen for DNMT-associated differentially expressed kinases.Small interfering RNA(siRNA)technology was used to interfere the expressions of screened extracellular regulated protein kinase(ERK1),homeodomain interacting protein kinase 2(HIPK2)and glycogen synthase kinase 3 beta(GSK3β)to obtain si-ERK1,si-HIPK2,and si-GSK3βcells.AML cell line HL-60 cultured at logarithmic growth phase were taken and divided into control group(no treatment),si-NC group(blank plasmid transfection),si-ERK1 group,si-HIPK2 group and si-GSK3βgroup.Cell counting kit(CCK-8)assay and flow cytometry were used to examine cell viability and apoptosis,respectively.Real-time quantitative PCR(RT-qPCR)and Western blot were used to detect the messenger RNA(mRNA)and protein expressions of HIPK2,GSK3β,ERK1,signal transducer and activator of transcription 3(STAT3),phosphorylated STAT3(P-STAT3),Janus kinase(JAK),phosphorylated JAK(P-JAK),phosphoinositide 3-kinase(PI3K),phosphorylated PI3K(P-PI3K),serine/threonine kinase Akt(AKT),and phosphorylated AKT(P-AKT).Results Bioinformatics analysis showed that there were statistically significant differences in the levels of cell cycle-dependent kinase 1(CDK1),casein kinase 2 alpha 1(CSNK2A1),mitogen-activated protein kinase 1(MAPK1),MAPK14,CDK4,ERK1,HIPK2,and GSK3βin AML patients between low-and high-DNMT1 groups(P<0.05).ERK1,HIPK2 and GSK3βwere finally selected for further study.Compared with the control group and si-NC group,the expression of HIPK2,GSK3β,and ERK1 protein in HL-60 cells with interference was decreased(P<0.001).When compared to control group and si-NC group,interfering the expressions of ERK1,HIPK2 or GSK3βin HL-60 cells led to a significant decrease in cell viability(P<0.05)and a significant increase

关 键 词：白血病 髓样 急性 DNA甲基转移酶1 生物信息学分析 细胞外调节蛋白激酶 同源结构域相互作用蛋白激酶2 糖原合酶激酶3Β JAK/STAT1通路 PI3K/AKT通路 

分 类 号：R733.71[医药卫生—肿瘤] Q811.4[医药卫生—临床医学]