Fig. 5

Direct targeting of SOCS6 and activation of JAK2/STAT3 signaling pathway by exosomal miR-106a-5p in macrophages. A. Venn plot showing the potential target genes predicted to bind with miR-106a-5p by five bioinformatics tools. B. The predicted binding sites of miR-106a-5p on the 3’UTR of SOCS6. C. In HEK 293T cells, dual-luciferase reporter gene assays were conducted using transfection with either wild or mutant SOCS6 3’-UTR plasma, along with miR-106a-5p mimics or inhibitor. The luciferase activity was detected 48 h post-transfection and normalized based on the ratio of firefly to renilla luciferase signals. D. 48 h following the transfection of miR-106a-5p mimics in Mφ, western blot was conducted to detect the expression of SOCS6 and the activation of the JAK2/STAT3 pathway. E. qRT-PCR was employed to detect the expression of SOCS6 in Mφ after treatment with different exosomes. F. 48 h following the co-culture of different exosomes in Mφ, western blot was conducted to detect the expression of SOCS6 and the activation of the JAK2/STAT3 pathway. G-H. Mφ were transfected with miR-106a-5p mimics alone or combined with SOCS6-OE. Flow cytometry was employed to assess the proportion of CD206⁺ macrophages (G). qRT-PCR was used to measure the expression of IL-10, CD206, CD163, and Arginase-1 (H). I-J. Mφ were treated with HCT 116 exosomes alone or concurrently transfected with SOCS6-OE. Flow cytometry was employed to determine the proportion of CD206⁺ macrophages (I). qRT-PCR was used to evaluate the expression of IL-10, CD206, CD163, and Arginase-1 (J). K-L. Mφ were differentiated into M2 macrophage using IL-4 (50 ng/mL) and IL-13 (50 ng/mL), followed by transfection with either miR-106a-5p inhibitor alone or combined with sh-SOCS6. Flow cytometry was utilized to assess the proportion of CD206⁺ macrophages (K). qRT-PCR was conducted to measure the expression of IL-10, CD206, CD163, and Arginase-1 (L). M. Mφ were transfected with miR-106a-5p mimics alone or combined with SOCS6-OE, followed by western blotting to detect SOCS6 expression and JAK2/STAT3 pathway activation. N. Mφ were treated with HCT 116 exosomes alone or concurrently transfected with SOCS6-OE, followed by western blotting to detect SOCS6 expression and JAK2/STAT3 pathway activation. O. Mφ were differentiated into M2 macrophages using IL-4 and IL-13, then transfected with miR-106a-5p inhibitor alone or combined with sh-SOCS6, followed by western blotting to detect SOCS6 expression and JAK2/STAT3 pathway activation. The data presented herein represent the outcomes of a minimum of three independent experiments and are depicted as the mean ± standard deviation (SD). ns = No significant difference, * P < 0.05, ** P < 0.01, *** P < 0.001