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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 lymphoblastic leukemia T lymphocytes (CCRF-CEM) and its effect on cell biology, and to observe the effect of miR-218 on target gene c-kit Expression of the impact of human leukemia treatment provides new methods and new strategies. Methods: The expression of miR-218 in peripheral blood T lymphocytes and CCRF-CEM was detected by quantitative real-time PCR (qRT-PCR). The expression of miR-218 in CCRF-CEM cells was detected by qRT-PCR 48 hours after transfection with miR-218 mimic in CCRF-CEM cells. The effect of miR-218 on the viability of CCRF-CEM cells was detected by MTT assay. The effect of miR-218 on the cell cycle and apoptosis of CCRF-CEM cells was analyzed by flow cytometry. C-kit was used as the target gene for miR-218 regulation by fluorescence reporter enzyme-linked enzyme system, and the effect of miR-218 on KIT protein expression in CCRF-CEM cells was detected by Western blot. Results: The results of qRT-PCR showed that the expression of miR-218 in CCRF-CEM cell line was significantly lower than that in normal human peripheral blood T lymphocytes (P <0.01). Compared with the control group, the miR-218 expression was significantly increased in miR-218 mimic cells transfected CCRF-CEM for 48 h (P <0.01). The results of MTT showed that miR-218 overexpression significantly decreased cell viability in CCRF-CEM cells (P <0.05). Flow cytometry analysis showed that the percentage of S phase cells in miR-218mimic transfected cells was significantly decreased (P <0.01), and the apoptosis of cells was significantly increased (P <0.01) after overexpression of miR-218. The results of luciferase reporter assay showed that the relative luciferase activity of transfected miR-218 mimic group was significantly lower than that of the control group (P <0.01). Western blot results showed that the expression of KIT protein was significantly decreased in miR-218 mimic cells transfected with CCRF-CEM compared with the control group (P <0.01). Conclusion: miR-218 is down-regulated in human acute lymphoblastic leukemia T lymphocytes (CCRF-CEM). MiR-218 negatively regulates the expression of KIT protein and inhibits the proliferation of CCRF-CEM cells and promotes apoptosis. Treatment strategies that enhance the expression of miR-218 are expected to benefit patients with leukemia.