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通过在体外将miR-223 duplex转染至粒系白血病细胞株(HL-60、NB4),观察其对粒系白血病细胞增殖作用的影响,来探讨miR-223对白血病细胞诱导分化及作用机理。以白血病HL-60、NB4细胞株为靶细胞,采用MTT法和半固体集落法观察不同浓度的miR-223对HL-60、NB4细胞增殖的影响;流式细胞术观察粒系细胞分化的表面标志(CD11b+、CD15+)表达;RT-PCR检测miR-223对c/EBPα、NFI-A转录因子表达的影响以及用免疫印迹反应观察相应蛋白的表达情况。结果表明:(1)低浓度的miR-223(10nM、20nM)均抑制HL-60、NB4细胞增殖(P<0.05或P<0.01),但在HL-60细胞中随着其浓度的增加(50nM、100nM),抑制作用与对照组相比无差异;而NB4细胞中随浓度增加抑制作用却没有增强(P<0.05)。半固体集落培养结果显示与MTT结果相一致。(2)流式细胞术检测用不同浓度的miR-223转染粒系细胞中发现,粒系细胞表面标志CD11b+表达在低浓度(10nM、20nM)下随其浓度升高而增加,但浓度增至50nM、100nM时表达有所下降;而CD15+在HL-60细胞中表达无显著差异,NB4细胞中表达趋势与CD11b+一致。(3)两细胞在miR-223作用前后提取总RNA分别进行RT-PCR检测显示,NB4细胞中NFI-A转录因子表达随浓度增加而降低,分别低于未经处理的0.6倍、2.0倍、1.1倍和1.5倍。c/EBPα基因表达随浓度增加而增加,分别是未经处理细胞的1.5倍、2.0倍、1.8倍和2.0倍。而在HL-60细胞中c/EBPα基因表达无明显差异,NFI-A仅在20nM浓度下表达下降差异显著,低于未经处理细胞的3.0倍。对NB4细胞中的NFI-A蛋白免疫印迹分析发现,经miR-223处理过的细胞中NFI-A蛋白含量随浓度增加而有所下降。结论:转染一定浓度miR-223至粒系白血病细胞中,能够抑制恶性细胞克隆增殖,其作用机制有可能通过竞争性抑制NFI-A转录因子表达,恢复与分化相关的c/EBPα转录因子的表达,来诱导粒系白血病细胞向成熟方向分化。
To investigate the effect of miR-223 on leukemic cell differentiation and its mechanism by in vitro transfection of miR-223 duplex into myeloid leukemia cell lines (HL-60, NB4) and observe its effect on the proliferation of myeloid leukemia cells. The effect of different concentrations of miR-223 on the proliferation of HL-60 and NB4 cells was observed by MTT assay and semi-solid colony assay using leukemia cells HL-60 and NB4 as target cells. Flow cytometry was used to observe the differentiation of granulosa cells (CD11b +, CD15 +). The effect of miR-223 on the expression of c / EBPα and NFI-A transcription factors was detected by RT-PCR and the corresponding protein expression was observed by immunoblotting. The results showed that: (1) Low concentrations of miR-223 (10 nM, 20 nM) inhibited the proliferation of HL-60 and NB4 cells (P <0.05 or P <0.01) 50nM, 100nM). There was no difference between the control group and the NB4 cells (P <0.05). Semi-solid colony culture results showed consistent with MTT results. (2) Flow cytometry detected with different concentrations of miR-223 transfected granulosa cells found that the expression of granulocyte-surface marker CD11b + at low concentrations (10nM, 20nM) increased with its concentration increased, but the concentration increased To 50nM and 100nM, while there was no significant difference in the expression of CD15 + in HL-60 cells. The expression of CD15 + in NB4 cells was consistent with that of CD11b +. (3) RT-PCR results showed that the expression of NFI-A transcription factor in NB4 cells decreased with the increase of concentration, which were 0.6 times and 2.0 times that of untreated cells, respectively. (3) 1.1 times and 1.5 times. The c / EBPα gene expression increased with increasing concentration, which was 1.5 times, 2.0 times, 1.8 times and 2.0 times that of untreated cells respectively. The expression of c / EBPα gene in HL-60 cells had no significant difference. The expression of NFI-A at 20nM concentration was significantly lower than that of untreated cells. Immunoblot analysis of NFI-A protein in NB4 cells revealed a decrease in NFI-A protein content with increasing concentrations in miR-223 treated cells. CONCLUSION: Transfection of a certain concentration of miR-223 into myeloid leukemia cells can inhibit the proliferation of malignant cell clones, and its mechanism may be through competitive inhibition of NFI-A transcription factor expression and recovery of differentiation-related c / EBPα transcription factor Expression, to induce myeloid leukemia cells to mature differentiation.