Let-7d inhibits intratumoral macrophage M2 polarization and subsequent tumor angiogenesis by targeting IL-13 and IL-10

Abstract Background: The microRNA let-7d has been reported to be a tumor suppressor in many types of cancer, including renal cell carcinoma (RCC). Tumor-associated macrophages are M2-polarized macrophages that can enhance tumor growth and angiogenesis in many human cancers. However, the role of let-7d in TAM-associated RCC tumor progression remains elusive.Methods: Human macrophage cell line THP-1 cells pretreated with phorbol myristate acetate were co-cultured with let-7d overexpressing RCC cells, the conditioned medium were obtained and its effect on human umbilical vein endothelial cells (HUVECs) was accessed in vitro. The phenotype of cocultured macrophages and macrophages in mouse models bearing let-7d overexpressing RCC cell derived xenograft were analyzed in vitro and in vivo using flow cytometry and quantitative real-time PCR.Results: First, we observed a strongly inverse correlation between let-7d expression and microvessel density in RCC tissues. Furthermore, the proliferation, migration, and tube formation of HUVECs was significantly inhibited by conditioned medium from a coculture system of the human macrophage THP-1 cell line with let-7d-overexpressing RCC cells. Moreover, the proportion of M2 macrophages was significantly lower in the group that was cocultured with let-7d-overexpressing RCC cells. Subcutaneous xenografts formed by the injection of let-7d-overexpressing RCC cells together with THP-1 cells resulted in a significant decrease in the M2 macrophage ratio and microvessel density compared with those formed by the injection of control RCC cells with THP-1 cells. In silico analysis revealed interleukin-10 (IL-10) and IL-13 as potential let-7d target genes. Consistently, the activities of the luciferase reporters containing the 3’-UTRs of the IL-10 and IL-13 genes were significantly inhibited by let-7d. Importantly, the addition of IL-10 and IL-13 counteracted the inhibitory effects of the conditioned medium from the coculture system of let-7d-overexpressing RCC cells in vitro. Additionally, the overexpression of IL-10 and IL-13 reversed the effects of let-7d on macrophage M2 polarization and tumor angiogenesis in vivo. Finally, the expression of IL-10 and IL-13 were inversely correlated with the expression of let-7d in RCC clinical specimens.Conclusions: These results suggest that let-7d may inhibit intratumoral macrophage M2 polarization and subsequent tumor angiogenesis at least in part by targeting IL-10 and IL-13.