miRNA-100低表达间充质干细胞对急性髓细胞性白血病细胞增殖、凋亡的影响及机制  

Effects and mechanism of low expression of miR-100 in mesenchymal stem cells on proliferation and apoptosis of acute myeloid leukemia cells

作　　者：缪华纬[1] 孔凡良[1] 蒋英俊[1] 王凤云[1] MIAO Huawei;KONG Fanliang;JIANG Yingjun;WANG Fengyun(The Second People′s Hospital of Hefei,Hefei 230011,China)

机构地区：[1]合肥市第二人民医院,合肥230011

出　　处：《山东医药》2020年第20期20-23,共4页Shandong Medical Journal

基　　金：安徽省自然科学基金项目(1908085QH359)。

摘　　要：目的观察miRNA-100低表达的间充质干细胞(MSC)对急性髓细胞性白血病细胞增殖、凋亡的影响,初步探讨其相关机制。方法MSC分别感染miR-100-NC和miR-100-inhibitor慢病毒,与人白血病细胞株K562共培养3 d后获取K562细胞,分别分为对照组和低表达组;采用MTT实验和平板克隆实验检测细胞增殖能力,采用流式细胞术检测细胞凋亡情况,Western blotting法检测细胞中的细胞周期蛋白细胞周期依赖性激酶4(CDK4)、细胞周期蛋白1(Cyclin D1)和凋亡相关蛋白Bcl-2样蛋白x(Bax)、cleaved Caspase-3。筛选出感染miR-100-NC和miR-100-inhibitor慢病毒的MSC中差异表达最明显的生长因子为骨形态发生蛋白4(BMP4),采用ELISA法检测K562细胞培养液中的BMP4。将K562细胞分为对照组、低表达组和实验组,对照组细胞与感染miR-100-NC慢病毒的MSC共培养,低表达组和实验组细胞与感染miR-100-inhibitor慢病毒的MSC共培养,实验组共培养1 d后加入BMP抑制剂Noggin蛋白;培养2 d后收集K562细胞,采用MTT实验和平板克隆实验检测增殖能力,流式细胞术检测凋亡细胞,Western blotting法检测CDK4、Cyclin D1、Bax、cleaved Caspase-3。结果低表达组细胞转染72 h细胞增殖能力及克隆形成数均高于对照组(P均<0.05);低表达组Cyclin D1、CDK4相对表达量高于对照组(P均<0.05);低表达组细胞凋亡率及Bax、cleaved Caspase-3相对表达量低于对照组(P均<0.05);低表达组培养液中BMP4水平高于对照组(P<0.05)。加入Noggin后,实验组共培养72 h时细胞增殖能力、克隆形成数及CDK4、Cyclin D1相对表达量均低于低表达组,细胞凋亡率和Bax、cleaved Caspase-3相对表达量高于低表达组(P均<0.05)。结论miR-100低表达的MSC可促进急性髓细胞性白血病细胞增殖、抑制其凋亡,可能是通过上调BMP4水平实现的。Objective To observe the effects of mesechymal stem cells(MSCs)with low expression of miR-100 on the proliferation and apoptosis of acute myoloid leukemia cells.Methods MSCs were infected with Lv-miR-100-NC and Lv-miR-100-inhibitor,namely control group and low-expression group,respectively.They were co-cultured with human leukemia cell line K562 for 3 days.Cell proliferation ability was detected by MTT assay and plate cloning assay,the apoptosis was examined by flow cytometry,and cyclin-dependent kinase 4(CDK4),Cyclin D1,apoptosis-related protein Bcl-2-associated X(BAX),and cleaved Caspase-3 were detected by Western blotting.Bone morphogenetic protein 4(BMP4)was identified as the most significantly differentially expressed growth factor in MSC infected with Lv-miR-100-NC and Lv-miR-100-inhibitor,and BMP4 concentration in K562 cells was detected by ELISA.K562 cells were then divided into the control group,low expression group and experimental group.The cells in the control group were co-cultured with MSCs infected with miR-100-NC lentivirus;the cells in the low expression group and experimental group were co-cultured with MSC infected with miR-100-inhibitor lentivirus.After 2 days of culture,K562 cells were collected.MTT assay and plate cloning assay were used to detect proliferation ability,flow cytometry was used to detect apoptotic cells,and CDK4,Cyclin D1,Bax,and cleaved Caspase-3 were detected by Western blotting.Results The proliferation ability and the number of clone formation were higher in the low expression group than in the control group after transfection for 72 h(both P<0.05).The relative expression levels of Cyclin D1 and CDK4 in the low expression group were higher than those in the control group(both P<0.05).The apoptosis rate and relative expression levels of Bax and cleaved Caspase-3 in the low expression group were lower than those in the control group(all P<0.05).Compared with the control group,mRNA sequencing and ELISA assay showed that BMP4 promoted the proliferation rather than apoptosis of

关 键 词：间充质干细胞 急性髓细胞性白血病 miRNA-100 细胞凋亡 骨形态发生蛋白4 

分 类 号：R733.7[医药卫生—肿瘤]