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 expression level of ARPP19 protein in THP-1 cells of miR-618 overexpression group was lower than that of the negative control group,while the expression levels of ARPP19 protein of peripheral blood monocytes of the healthy people in both groups were similar.Conclusion miR-618 can inhibit the cell proliferation and promote apoptosis of THP-1 cells by inhibiting the expression of of THP-1 cells ARPP19 in acute monocyte leukemia.
作者
李峰
王刚
步鹏
杨林花
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)
出处
《白血病．淋巴瘤》
CAS
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)。
