Supplementary Material for: MicroRNA-126 Deficiency Affects the Development of Thymus CD4⁺ Single-Positive Cells through Elevating IRS-1

Background: MicroRNA-126 (miR-126), a distinct miRNA family member, has been reported to be involved in the development and function of some types of immune cells. However, the potential role of miR-126 in the development of CD4+ T cells remains to be elucidated. Objectives: To investigate the potential role of miR-126 in the development of CD4+ T cells in the thymus and explore its significance. Methods: The relative expression level of miR-126 in thymus CD4+ single-positive (SP) cells was detected by Real-Time PCR assay. The possible change in thymus tissue was assessed by histopathology. The total cell number of thymocytes and the expression of activation-associated molecules including CD62L, CD69, and CD44, as well as proliferation-associated nuclear antigen Ki-67, in CD4+ SP cells were assessed by flow cytometric analysis. The expression of IRS-1 and related signaling pathways including Akt and Erk were determined by flow cytometric analysis. Results: Compared with that in wild-type (WT) mice, the total cell number of thymocytes in miR-126 knockdown (KD) mice increased significantly. Moreover, the proportion and absolute cell number of thymic CD4+ SP cells decreased significantly in miR-126 KD mice. Further analysis showed that the frequencies of activation-associated molecules including CD62L, CD69, and CD44, as well as proliferation-associated nuclear antigen Ki-67 in CD4+ SP cells also changed significantly, respectively. Mechanism aspect, the expression level of IRS-1, a putative target of miR-126, increased significantly in CD4+ SP cells in miR-126 KD mice. Moreover, the expression levels of the signaling molecules phosphorylated (p)-Akt and p-Erk also changed significantly. Conclusions: Our work is the first to reveal a previously unknown role of miR-126 in the development of CD4+ SP cells in the thymus, which might ultimately benefit studies on development of thymocytes.背景：微小RNA-126（MicroRNA-126，miR-126）是一类独特的微小RNA家族成员，已有研究证实其参与多种免疫细胞的发育与功能调控。但miR-126在CD4+ T细胞发育过程中的潜在作用仍有待阐明。 研究目标：探讨miR-126在胸腺CD4+ T细胞发育中的潜在作用，并揭示其生物学意义。 实验方法：采用实时荧光定量PCR（Real-Time PCR）技术检测胸腺CD4+单阳性（single-positive，SP）细胞中miR-126的相对表达水平；通过组织病理学检查评估胸腺组织的形态学变化；采用流式细胞术分析胸腺细胞总数，以及CD4+ SP细胞中CD62L、CD69、CD44等活化相关分子与增殖相关核抗原Ki-67的表达水平；同时通过流式细胞术检测胰岛素受体底物1（IRS-1）及Akt、Erk相关信号通路分子的表达水平。 实验结果：与野生型（wild-type，WT）小鼠相比，miR-126敲低（knockdown，KD）小鼠的胸腺细胞总数显著升高。此外，miR-126 KD小鼠的胸腺CD4+ SP细胞比例与绝对计数均显著降低。进一步分析显示，CD4+ SP细胞中CD62L、CD69、CD44等活化相关分子及增殖相关核抗原Ki-67的表达频率均发生显著改变。机制研究方面，miR-126的潜在靶基因IRS-1在miR-126 KD小鼠的CD4+ SP细胞中表达水平显著升高；同时，磷酸化Akt（p-Akt）与磷酸化Erk（p-Erk）等信号分子的表达水平亦发生显著改变。 结论：本研究首次揭示了miR-126在胸腺CD4+ SP细胞发育中此前未被报道的作用，该发现可为胸腺细胞发育相关研究提供新的参考依据。