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.