Cbl-b通过HOXA10调控miR-99a和miR-125b参与CD4<sup>+</sup>T细胞活化的机制研究-东南大学学报医学版

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

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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