miRNA-618对急性单核细胞白血病THP-1细胞增殖和凋亡调控的研究  

Regulation of miRNA⁃618 on the proliferation and apoptosis of acute monocyte leukemia THP⁃1 cells

作　　者：李峰 王刚[1] 步鹏[2] 杨林花[1] Li Feng;Wang Gang;Bu Peng;Yang Linhua(Department of Hematology,the Second Hospital of Shanxi Medical University,Taiyuan 030001,China;Department of Pathology,Shanxi Provincial Cancer Hospital,Taiyuan 030013,China)

机构地区：[1]山西医科大学第二医院血液内科,太原030001 [2]山西省肿瘤医院病理科,太原030013

出　　处：《白血病．淋巴瘤》2021年第3期156-160,共5页Journal of Leukemia & Lymphoma

基　　金：山西省应用基础研究项目(201801D121303);山西省肿瘤医院博士基金(2017A07);山西省肿瘤医院科研创新团队建设项目(202003)。

摘　　要：目的探讨miRNA-618(miR-618)对急性单核细胞白血病THP-1细胞增殖和凋亡的影响。方法采用实时荧光定量聚合酶链反应(PCR)检测miR-618在THP-1细胞和健康人外周血分离的单核细胞中的相对表达量。构建过表达miR-618质粒载体,以空载体作为阴性对照,将二者分别转染THP-1细胞,设定为miR-618过表达组和阴性对照组。采用CCK-8法和流式细胞术分别检测各组THP-1细胞增殖和凋亡情况。采用TargetScan软件预测miR-618靶基因,通过荧光素酶报告基因实验对其进行验证。采用蛋白质印迹法检测miR-618过表达组或阴性对照组的THP-1细胞和健康人外周血单核细胞中预测的miR-618靶基因蛋白表达的水平。结果PCR结果显示,与健康人外周血分离的单核细胞相比,THP-1细胞中miR-618表达量低(P<0.05)。CCK-8法检测结果显示,与阴性对照组比较,miR-618过表达组THP-1细胞增殖能力降低(转染0、24、48、72 h细胞吸光度值:0.20±0.03比0.20±0.03、0.28±0.02比0.35±0.03、0.34±0.03比0.43±0.04、0.39±0.02比0.53±0.05,均P<0.05),细胞晚期凋亡率升高[(27.1±0.1)%比(14.9±0.1)%,t=2.13,P=0.03]。TargetScan软件预测miR-618靶基因为ARPP19。荧光素酶报告基因实验结果显示,转染野生型ARPP19基因质粒+miR-618基因质粒组的THP-1细胞相对荧光素酶活性均高于空白对照组和转染野生型ARPP19基因质粒+miR-618空载质粒组(0.170±0.003比0.100±0.004、0.100±0.001,均P<0.05)。蛋白质印迹法检测结果显示,miR-618过表达组THP-1细胞ARPP19蛋白表达水平低于阴性对照组,而两组健康人外周血单核细胞中ARPP19蛋白表达水平相近。结论miR-618可能通过抑制急性单核细胞白血病THP-1细胞ARPP19的表达而抑制细胞增殖、促进细胞凋亡。Objective To investigate the effect of miRNA-618(miR-618)on the cell proliferation and apoptosis of acute monocyte leukemia THP-1 cells.Methods Real-time polymerase chain reaction(PCR)was used to detect the relative expression level of miR-618 in THP-1 cells and monocytes isolated from peripheral blood of the healthy people.Overexpression of miR-618 plasimid vector was constructed and empty vector was treated as the negative control;and then the two vectors were transfected with THP-1 cells;finally,miR-618 overexpression group and negative control group were set.THP-1 cell proliferation and apoptosis of both groups were detected by using CCK-8 method and flow cytometry,respectively.TargetScan was used to predict the target gene of miR-618 and it was verified by using luciferase reporter assay.Western blot was used to detect the protein levels of THP-1 cells in miR-618 overexpression group and negative control group,and predicted miR-618 target gene in peripheral blood monocytes of the healthy people.Results PCR showed that the expression level of miR-618 was lower in THP-1 cells compared with that in monocytes isolated from peripheral blood of the healthy people(P<0.05).CCK-8 assay showed that compared with the negative control group,the proliferation ability of THP-1 cells in miR-618 overexpression group was decreased(the absorbance values at 0,24,48 and 72 h after transfection:0.20±0.03 vs.0.20±0.03,0.28±0.02 vs.0.35±0.03,0.34±0.03 vs.0.43±0.04,0.39±0.02 vs.0.53±0.05,all P<0.05),and the late apoptosis rate was increased[(27.1±0.1)%vs.(14.9±0.1)%,t=2.13,P=0.03].The target gene of miR-618 was ARPP19 predicted by using TargetScan software.Luciferase reporter assay showed that the relative luciferase activity of THP-1 cells in group transfected with wild-type ARPP19 gene plasmid+miR-618 gene plasmid was higher than that in the blank control group and group transfected with wild-type ARPP19 gene plasmid+miR-618 empty vector(0.170±0.003 vs.0.100±0.004,0.100±0.001,all P<0.05).Western blot indicated the ex

关 键 词：白血病 单核细胞 急性 微RNAS 细胞增殖 细胞凋亡 miRNA-618 

分 类 号：R733.71[医药卫生—肿瘤]