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

The mechanism of Cbl- b participates in the activation of CD4^+ T cell through HOXA10 which regulates miR- 99a and miR- 125b

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.