miR-181b-5p通过靶向结合PAX9促进人急性髓细胞性白血病细胞增殖并诱导其凋亡  

miR-181b-5p promotes cell proliferation and induces apoptosis in human acute myeloid leukemia by targeting PAX9

作　　者：李斌 陶千山[2] 胡雪莹 李坦 鲍扬漪 LI Bin;TAO Qianshan;HU Xueying;LI Tan;BAO Yangyi(Department of Hematology,Third Affiliated Hospital of Anhui Medical University/Hefei First People's Hospital,Second Affiliated Hospital of Anhui Medical University,Hefei 230001,China;Department of Hematology,Second Affiliated Hospital of Anhui Medical University,Hefei 230001,China)

机构地区：[1]安徽医科大学第三附属医院/合肥市第一人民医院血液科,安徽合肥230001 [2]安徽医科大学第二附属医院血液科,安徽合肥230001

出　　处：《细胞与分子免疫学杂志》2023年第12期1074-1082,共9页Chinese Journal of Cellular and Molecular Immunology

基　　金：2019年合肥市应用医学研究项目(2019172)。

摘　　要：目的 探究miR-181b-5p通过靶向结合配对盒基因9(PAX9)对急性髓细胞性白血病(AML)细胞增殖和凋亡的影响。方法 分别用基因表达谱交互分析(GEPIA)数据库和肿瘤基因组图谱(TCGA)数据库分析AML患者中PAX9的表达水平和预后的关系。在Kasumi-1细胞和AML5细胞中转染空载体(Vector组)或PAX9(PAX9组),CCK-8法检测细胞的增殖活性,流式细胞术检测细胞周期和细胞凋亡,Western blot法检测细胞周期蛋白依赖激酶2(CDK2)和细胞周期蛋白B1(CCNB1)、B细胞淋巴瘤因子2(Bcl2)、 Bcl2相关X蛋白(BAX)的表达。生物信息学分析预测PAX9靶作用的微小RNA(miRNA)、荧光素酶报告系统验证其靶向作用,实时定量PCR检测PAX9 mRNA水平,Western blot法检测PAX9蛋白表达。在Kasumi-1细胞和AML5细胞中转染miR-NC(miR-NC组)或miR-181b-5p(miR-181b-5p组),在miR-181b-5p组细胞中进一步转染PAX9(miR-181b-5p联合PAX9组),采用前述方法分别检测细胞增殖、细胞周期和细胞凋亡。结果 GEPIA和TCGA数据库显示AML患者PAX9呈低表达趋势并与患者不良预后相关。在Kasumi-1、 AML5细胞中,与Vector组相比,PAX9组细胞增殖活力、 S期细胞百分比、 CDK2、 CCNB1和Bcl2蛋白表达降低,G0/G1期细胞百分比、细胞凋亡率、 BAX蛋白表达升高。双荧光素酶报告基因验证PAX9是miR-181b-5p的靶基因。与miR-NC组相比,miR-181b-5p组细胞增殖活力、 S期细胞百分比、 CDK2、 CCNB1和Bcl2蛋白表达升高,G0/G1期细胞百分比、细胞凋亡率、 BAX蛋白表达降低。与miR-181b-5p组相比,miR-181b-5p联合PAX9组细胞增殖活力、 S期细胞百分比、 CDK2、 CCNB1和Bcl2蛋白表达降低,G0/G1期细胞百分比、细胞凋亡、 BAX蛋白表达升高。结论 miR-181b-5p靶向抑制PAX9表达促进AML细胞增殖、延缓细胞凋亡。ObjectiveTo investigate the effects of miR-181b-5p on cells proliferation and apoptosis in acute myeloid leukemia(AML)bytargetingpairedbox9(PAX9).Methods Therelationship between expression level of PAX9 and prognosis in AML patients was analyzed by gene expression profiling interactive analysis(GEPIA)database and The Cancer Genome Atlas(TCGA)database.Kasumi-1 and AML5 cells were transfected with empty vector(Vector group)or PAX9(PAX9 group).The proliferation activity was detected by CCK-8 assay,and cells cycle and apoptosis were detected by flow cytometry.Expressions of cyclin-dependent kinase 2(CDK2),cyclin B1(CCNB1),B-cell lymphoma 2(Bcl2)and Bcl2-associated X protein(BAX)were detected by Western blot analysis.The targeted microRNA(miRNA)by PAX9 was predicted by bioinformatics analysis,and the targeted effect was verified by luciferase reporter assay.The level of PAX9 mRNA was detected by real-time quantitative PCR,and expression of PAX9 protein was detected by Western blot analysis.Kasumi-1 and AML5 cells were transfected with miR-NC(miR-NC group)or miR-181b-5p(miR-181b-5p group).The cells were further transfected with PAX9(miR-181b-5p combined with PAX9 group)in miR-181b-5p group.The proliferation,cycle and apoptosis of cells were detected by the above methods.Results GEPIA and TCGA databases showed that the expression of PAX9 was down-regulated in AML patients,which was correlated with poor prognosis.In Kasumi-1 and AML5 cells,compared with Vector group,proliferation activity of cells,percentage of cells in S phase,and expressions of CDK2,CCNB1 and Bcl2 proteins were decreased,while percentage of cells in GO/G1 phase,apoptosis rate and the expression of BAX protein were increased in PAX9 group.It was confirmed by double luciferase reporter assay that PAX9 was the target gene of miR-181b-5p.Compared with miR-NC group,proliferation activity of cells,percentage of cells in S phase,and expressions of CDK2,CCNB1 and Bcl2 proteins were increased,while percentage of cells in GO/G1 phase,apoptosis rate and the expre

关 键 词：急性髓细胞样白血病(AML) 配对盒基因9(PAX9) miR-181b-5p 细胞增殖 细胞凋亡 细胞周期 

分 类 号：R733.71[医药卫生—肿瘤] S853.53[医药卫生—临床医学] R714.254[农业科学—临床兽医学]