摘要
目的:检测microRNA-218(miR-218)在人急性淋巴细胞白血病T淋巴细胞(CCRF-CEM)中的表达情况和其对细胞生物学特性的影响,并观察miR-218对靶基因c-kit表达的影响,为人白血病治疗提供新方法和新策略。方法:利用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)检测miR-218在正常人外周血T淋巴细胞和CCRF-CEM中的表达情况。在CCRF-CEM细胞中转染miR-218 mimic后48 h,qRT-PCR检测CCRF-CEM细胞中miR-218表达变化;MTT检测miR-218对CCRF-CEM细胞活力的影响。流式细胞仪分析miR-218对细胞CCRF-CEM的细胞周期和凋亡的影响。利用荧光报告酶系统检验c-kit是miR-218调控靶基因,并采用Western blot方法检测miR-218对CCRF-CEM细胞中KIT蛋白表达的影响。结果:qRT-PCR检测结果显示:与正常人外周血T淋巴细胞相比,miR-218在CCRF-CEM细胞系中的表达显著下降(P<0.01)。与对照组相比,在CCRF-CEM中转染miR-218 mimic 48 h后,细胞中miR-218的表达显著上升(P<0.01)。MTT结果显示:在CCRF-CEM细胞中过表达miR-218后,细胞活力显著下降(P<0.05)。流式细胞仪分析结果显示:过表达miR-218后,miR-218mimic转染组细胞的S期细胞比例明显下降(P<0.01);细胞的凋亡显著增加(P<0.01)。荧光素酶报告基因系统检测结果显示:与对照组相比,转染miR-218 mimic组的相对荧光素酶活力显著降低(P<0.01)。Western blot检测结果显示:与对照组相比,在CCRF-CEM中转染miR-218 mimic 48 h后,细胞中KIT蛋白的表达显著下降(P<0.01)。结论:miR-218在人急性淋巴细胞白血病T淋巴细胞(CCRF-CEM)中表达下调,miR-218可负性调节KIT蛋白的表达,并抑制CCRF-CEM细胞增殖,促进凋亡。增强miR-218表达的治疗策略有望使白血病患者受益。
Objective： To detect the expression of microRNA-218（ miR-218） in human acute lymphocyte leukemia（ ALL） T lymphocytes（ CCRF-CEM）,explore its effects on the biological features of CCRF-CEM cells and the expression of its target gene c-kit,so as to provide new insights for leukemia treatment. Methods： Using the quantitative real-time polymerase chain reaction（ qRTPCR）,we detected the expression of miR-218 in the normal peripheral blood T lymphocytes and CCRF-CEM cells. Forty-eight hours after the miR-218 mimic was transfected into the CCRF-CEM cells,the expression of miR-218 in the CCRF-CEM cells was detected by qRT-PCR. The effect of miR-218 on the CCRF-CEM cell viability was detected using MTT. The effect of miR-218 on the proliferation and apoptosis of CCRF-CEM cell was analyzed using flow cytometry. c-kit gene was identified to be a target gene of miR-218 by luciferase reporter enzyme system,and the effect of miR-218 on the expression of KIT protein in cells were determined using Western blot. Results： As shown by qRT-PCR,compared with that in the normal peripheral blood T lymphocytes,the expressions of miR-218 in ALL T lymphocytes cell lines were significantly decreased（ P 〈0. 01）. Compared with the control group,the expression of miR-218 increase significantly in CCRF-CEM cells transfected with miR-218 mimic for 48 hours（ P 〈0. 01）. MTT showed that the cell viability decreased significantly after the over-expression of miR-218 in the CCRF-CEM cells（ P 〈0. 05）. Flow cytometry showed that the Sphase fraction significantly declined after the over-expression of miR-218（ P 〈0. 01）,and meanwhile the apoptosis of cells also significantly increased（ P 〈0. 01）. Luciferase reporter gene assay showed that,compared with the control group,the relative luciferase activity significantly declined in the miR-218 mimic transfection group（ P 〈0. 01）. Compared with the control group,the expression of KIT protein in the CCRF-CEM cells transfected with miR-218 mimic for 48 h
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2015年第1期103-108,共6页
Chinese Journal of Immunology
基金
江苏省卫生厅面上科研课题(H201422)
江苏省南通市市级科技计划项目(HS2013015)
南通大学附属医院科技项目(TDFzh2014015)资助
