MicroRNA-223在淋巴细胞白血病原代细胞中表达及作用机制的研究

本研究旨在探讨急性淋巴细胞白血病（ALL）、慢性淋巴细胞白血病（CLL）原代细胞中MicroRNA-223与LMO2基因的表达水平及MicroRNA-223的作用机制。应用人淋巴细胞分离液分选ALL、CLL患者及正常人骨髓中淋巴细胞,在ALL、CLL患者骨髓淋巴细胞中转染MicroRNA-223的类似物靶向升高细胞中MicroRNA-223的表达,在正常人骨髓淋巴细胞中转染MicroRNA-223抑制物靶向敲低细胞中MicroRNA-223的表达。转染后培养72 h,用RT-PCR方法检测转染前后MicroRNA-223和LMO2表达量及其相关性,并用流式细胞术进一步观察细胞凋亡和细胞周期的变化。结果表明,转染MicroRNA-223类似物前,ALL、CLL患者中MicroRNA-223表达水平为（433.11±144.88）,LMO2水平为（807.10±238.41）,正常人转染MicroRNA-223抑制物前,MicroRNA-223表达水平为（949.59±267.39）,LMO2的表达为（455.32±176.83）;MicrRNA-223的表达在正常人中明显高于ALL、CLL患者（P〈0.05）,而LMO2的表达在正常人中明显低于ALL、CLL患者（P〈0.05）。转染后,ALL、CLL患者中MicroRNA-223表达明显增加（571.86±142.00）（P〈0.05）,而LMO2表达明显减少（651.97±230.12）（P〈0.05）;在正常人中MicroRNA-223表达明显降低（646.32±172.93）（P〈0.05）,LMO2的表达明显升高（541.27±158.86）（P〈0.05）。转染后细胞周期及细胞凋亡率的变化表现为,在ALL、CLL患者转染前细胞周期G1/G2细胞比例为（94.75±3.15）%,S期为（5.14±3.12）%;转染后G1/G2细胞比例明显增加（97.03±2.08）%（P〈0.05）,在S期明显减少（2.97±2.08）%（P〈0.05）;转染前细胞凋亡率为（54.47±8.72）%,转染后为（60.48±8.81）%,后者明显增加（P〈0.05）。正常人转染前细胞周期G1/G2细胞比例是（96.73±2.26）%,S期是（3.25±2.26）%;转染后G1/G2细胞比例明显减少（94.55±2.77）%（P〈0.05）,在S期明显增加（5.45±2.77）%（P〈0.05）,细胞凋亡率为（59.02±10.20）%,转染后明显减少（51.96±10.20）%（P〈0.05）。结论：淋巴细胞白血病原代细胞中MicroRNA-223表达降低,LMO2表达增高,导致淋巴细胞增殖周期及凋亡异常,这可能是淋巴细胞白血病的发病机制之一。

Expression of MicroRNA-223 in Lymphocytic Leukemia Cells and Its Action Mechanism

This study was aimed to investigate the expression level and mechanism of microRNA-223 and LM02 in acute lymphoblastic leukemia（ALL） and chronic lymphocytic leukemia（CLL） cells and the mechnism. MicroRNA-223 mimics was transfected to increase the expression of MicroRNA-223 in the lymphocytes sorted by ficoll separation from the bone marrow mononuclear cells （BMMNC） of ALL and CLL patients. MicroRNA-223 inhibitor was transfected to decrease the expression of the MicroRNA-223 in the lymphocytes of normal controls. Then the expression of the MicroR- NA-223 and LM02 in transfected lymphocytes before and after cultivating for 72 hours were detecfed by RT-PCR, the apoptosis and cell cycle of these cells were measured by flow cytometery. The results indicated that before the transfec tion, the expression of MicroRNA-223 in ALL and CLL cells was （433.11 ± 144.88）, which was significantly lower than that in the norinal lymphocyte（949.59 ± 267.39 ） ; the expression of LM02 was （ 807.10 ± 238.41 ）, which wassignificantly higher than that in the normal lymphocytes （455.32 ± 176.83 ） （P 〈 0.05 ） ; after the transfection, the expr- eseion of MicroRNA-223 was （571.86± 142.00） in ALL and CLL cells, which was significantly higher than that before transfection （P 〈0.05）, but the expression of LM02 was significantly lower than that before transfection （651.97±230.12 ） （ P 〈 0.05 ） ; in the normal control the expreseion of MicroRNA-223 obviously decreased （ 646.32 ± 172.93 ） （ P 〈 0.05 ）, the expression of LM02 was significantly increased （ 541.27 ± 158.86.2 ） （ P 〈 0.05 ）. After transfection, the cell cycle G1/G2 phase and apoptosis changed in ALL and CLL cells. Before transfection the cell ratio in cell cycle GiG2 phase was （94.75 ±3.15）%, the cell ratio in S phase was （5.14 ±3.12）% ; after transfection the cell ratio in cell cycle G/G2 phase was （97.03 ± 2.08 ）% and obviously increased（ P 〈 0.05 ）, the cell ratio in S phase was （2.97 ± 2.08 ） % and significantly decreased （ P 〈 0.05 ）. Before transfection the apoptosis rate was （ 54.47 ± 8.72 ） %, and obviously was higher than that after transfection （60.48 ± 8.81 ） %. And in the normal control, the cell ratio in G1/G2 phase was significantly higher than that after transfection [ （96.73 ±2.26） %, （94.55 ±2.77） %, P 〈0.05） ], and the cell ratio in S phase was significantly increased[ （3.25 ± 2.26） % , （5.45 ± 2.77 ） % （ P 〈0.05 ） 1. The apoptotic rate in the ALL and CLL patients was significantly higher than that after the transfection [ （ 54.47 ± 8.72 ） % vs （ 60.48 ± 8.81 ） %, respectively （ P 〈 0.05 ） ]. The apoptotic rate in the namol control was significantly lower than that after the transfection [ （59.02 ± 10. 20 ） % , （51.96 ± 10.20） % , respectively （P 〈 0.05 ） 1. It is concluded that the expression of MicroRNA-223 decreases, and the expression of LM02 inceases in lymphocytic leukemia cells which leads to the lym phocytes over-proliferation and abnormal apoptosis, thus may be one of pathogenesis in lymphocytic leukemia.