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. 2022 May 12;12:808888. doi: 10.3389/fonc.2022.808888

Figure 5.

Figure 5

RCC-derived exosomal circSAFB2 induces M2 macrophage polarization through the miR-620/JAK1/STAT3 axis. (A, B) Schematic representation of the 3′- ‘UTR’ of JAK1 with the predicted target site for miR-620. The mutant site of JAK1 3′-UTR is indicated (without line); (C) Luciferase reporter analysis was performed to examine the binging capacity between miR-620 and JAK1. Reporter constructs containing JAK1wt or JAK1mut at the predicted miR-620 target sequences were co-transfected into macrophages, along with miR-620 mimics or miR-NC; (D, E) Western blotting and densitometric analysis of JAK1 expression in macrophages after transfection with miR-NC or miR-620 mimics; (F, G) Western blotting and densitometric analysis of JAK1 and STAT3 expression in macrophages after transfection with JAK1 siRNA; (H–K). (H, I) Western blotting and densitometric analysis value of JAK1 and STAT3 expression in macrophages after treatment with 769-P-derived exosomes (Exo-769-P), along with miR-NC or miR-620 mimics. (J, K) Western blotting and densitometric analysis value of JAK1 and STAT3 expression in macrophages after treatment with ACHN-derived exosomes (Exo-ACHN), along with miR-NC or miR-620 mimics; (L). qRT-PCR analysis of M2 macrophages (IL-1RA, CD163, CD206 and CCL18) after indicated treatment (Exo-769-P, Exo-769-P + si-circSAFB2, Exo-769-P + si-circSAFB2 pool + miR-620 int, Exo-769-P + si-circSAFB2 pool + miR-620 int + si-JAK1); (M). qRT-PCR analysis of M1 macrophages (TNF-α, IL-1β and MCP-1) after indicated treatment; (N). ELISA analysis of cytokine released from M2 macrophages (IL-1RA and CCL18) and M1 macrophages (TNF-α and IL-1β). All data indicate mean ± SD; n=3, **P<0.01, ***P<0.001. ns, no significance.