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通过在体外将miR-223 duplex转染至红白血病细胞株(HEL),观察其对HEL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HEL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HEL细胞增殖的影响;流式细胞术观察红系细胞分化的表面标志(CD71+、GPA+)表达;RT-PCR检测miR-223对LMO2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:低浓度的miR-223(10nM、20nM)抑制HEL细胞增殖,且随浓度增加(50nM、100nM)时,抑制作用更明显(P<0.05或P<0.01)。半固体集落培养结果显示与MTT结果相一致。流式细胞术检测用不同浓度的miR-223转染HEL细胞中发现红系细胞表面标志GPA+表达随其浓度升高而增加,而CD71+在细胞中表达无显著差异。在miR-223作用前后提取RNA进行RT-PCR检测显示,LMO2基因表达随浓度增加而降低。对LMO2蛋白免疫印迹分析发现,经miR-223处理过的细胞中LMO2蛋白含量随浓度增加逐渐下降。结论:转染一定浓度miR-223至红白血病HEL细胞中,能够抑制细胞增殖,其作用机制有可能通过竞争性抑制LMO2转录因子表达,恢复与分化相关的转录因子的表达,来诱导红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 transfecting miR-223 duplex into erythroleukemia cell line (HEL) in vitro and observing its effect on the proliferation of HEL cells. The effect of different concentrations of miR-223 on the proliferation of HEL cells was observed by MTT assay and semi-solid colony assay. The expression of CD71 + and GPA + on erythroid cells was observed by flow cytometry. The effect of miR-223 on the expression of LMO2 transcription factor was detected by RT-PCR and the corresponding protein expression was observed by Western blotting. The results showed that low concentration of miR-223 (10 nM, 20 nM) inhibited the proliferation of HEL cells, and the inhibitory effect was more obvious with increasing concentration (50 nM, 100 nM) (P <0.05 or P <0.01). Semi-solid colony culture results showed consistent with MTT results. Flow cytometry showed that the expression of erythroid cell surface marker GPA + increased with increasing concentrations of miR-223 transfected HEL cells, while there was no significant difference in the expression of CD71 +. The RNA extracted before and after the miR-223 treatment showed that the expression of LMO2 gene decreased with the increase of the concentration. Western blot analysis of LMO2 protein revealed that the concentration of LMO2 protein in the miR-223-treated cells decreased gradually with increasing concentration. CONCLUSION: Transfection of a certain concentration of miR-223 into human erythroleukemia HEL cells can inhibit the proliferation of human erythroleukemia cells by inhibiting the expression of LMO2 transcription factor and restoring the expression of differentiation-related transcription factors Differentiate to mature direction.