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Cbl-b通过HOXA10调控miR-99a和miR-125b参与CD4+T细胞活化的机制研究
作者:唐玉旭  杨燚  张进平 
单位:苏州大学生物医学研究院 张进平教授课题组, 江苏 苏州 215123
关键词:CD4+T细胞 卡西塔斯B细胞淋巴瘤谱系b 同源异型盒蛋白A10 微小RNA-99a 微小RNA-125b 
分类号:R392.1
出版年·卷·期(页码):2019·38·第六期(954-959)
摘要:

目的:探讨卡西塔斯B细胞淋巴瘤谱系b (Cbl-b)调控CD4+T细胞活化的机制。方法:用CCK8试剂盒检测过表达微小RNA (miR)-99a和miR-125b的小鼠淋巴瘤细胞(EL4)细胞增殖活性。取抗CD3和抗CD28抗体活化野生型(WT)和Cbl-b缺失型(Cbl-b-/-) CD4+T细胞,激光共聚焦观察WT和Cbl-b-/- CD4+T细胞中同源异型盒蛋白A10(HOXA10)的入核情况。用双荧光素酶报告系统检测HOXA10对miR-99a和miR-125b的表达调控作用。用蛋白质印迹法和双荧光素酶报告系统检测miR-99a和miR-125b对靶基因的表达水平影响。结果:在Cbl-b-/-CD4+T细胞中,HOXA10在抗CD3抗体单独刺激时即可入核。HOXA10入核后促进miR-99a和miR-125b表达,而过表达miR-99a和miR-125b则抑制蛋白激酶B2和3(AKT2、AKT3)、磷酸肌醇-3-激酶催化亚基A和D (PIK3CA、PIK3CD)的表达。结论:Cbl-b通过阻止HOXA10核转位来抑制miR-99a和miR-125b表达,导致AKT2、AKT3、PIK3CA、PIK3CD (AKT/PI3K)表达增加,最终抑制CD4+T细胞活化。

Objective: To explore the mechanism of Cbl-b in regulating the activation of CD4+ T cell. Methods: The proliferation activity of EL4 cells which overexpressed miR-99a and miR-125b was detected with CCK8 kit. Anti-CD3 or/and anti-CD28 antibodies were used to activated CD4+T cells which were purified from WT or Cbl-b-/- mice, then confocal microscopy was used to observe the translocation into nuclear of (homeobox protein A 10, HOXA10) in WT or Cbl-b-/- CD4+T cells. Dual-luciferase assay was used to detect the effect of HOXA10 on expression of miR-99a and miR-125b. The effect of miR-99a and miR-125b on the expression level of its target genes were evaluated by Western blot and dual-luciferase assay. Results: HOXA10 translocated into nuclear while the Cbl-b-/- CD4+T cells were activated by anti-CD3 antibody only. HOXA10 increased the expression of miR-99a and miR-125b while translocating into nuclear. Overexpression of miR-99a and miR-125b inhibited the expression of protein kinase B2 and 3, phosphoinositol 3-kinase catalytic subunits A and D.Conclusion: Cbl-b inhibits the expression of miR-99a and miR-125b through preventing the nuclear translocation of HOXA10, which results in the increased expression of AKT/PI3K, finally inhibits CD4+T cell activation.

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