Inhibition of let-7b-5p contributes to an anti-tumorigenic macrophage phenotype through the SOCS1/STAT pathway in prostate cancer
Authors: Rong Jiping , Xu Lu , Hu Yinying , Liu Fan , Yu Yanrong , Guo Hongyan , Ni Xudong , Huang Yanqin , Zhao Lin , wang zhigang 
Published: September 2020

Abstract
BackgroundDysfunction of microRNAs (miRNAs) is a major cause of aberrant expression of inflammatory cytokines and contributes to macrophage polarization. Proinflammatory M1 macrophages promote T helper (Th) 1 responses and show tumoricidal activity, whereas M2 macrophages display regulatory functions in tissue repair and remodeling and promote Th2 immune responses. Previous studies have shown that miRNA let-7 is associated with cellular differentiation and that the expression of let-7b-5p is significantly augmented in M2 macrophages. However, the mechanism by which let-7b-5p regulates macrophage differentiation in prostate cancer (PCa) remains largely unknown.MethodsHuman macrophages were induced by blood monocytes from healthy male donors, and M1 macrophages were polarized by stimulating them overnight with 100 ng/ml of lipopolysaccharides and 100 ng/ml of IFN-γ. Conditioned medium from PC-3 cells was used to induce prostatic macrophages (M-CMs) in vitro, and we then transfected let-7b-5p mimics or inhibitors into M1 and M-CMs for 72 hours. The expression of cluster of differentiation 206 (CD206) in each group was detected with the High- Throughput Connotation of Imaging System. We used quantitative real-time polymerase chain reaction (qRT-PCR) to examine the expression of the inflammatory cytokines IL-10, IL-12, IL-13, TNF-alpha, and let-7b in macrophages. SOCS1 protein levels were evaluated by ELISA, and the phosphorylation difference in STAT family member proteins was analyzed using CST signal-pathway chip. Phagocytosis by macrophages and the effect of macrophages on the proliferation of prostate cancer PC-3 cells were evaluated with phagocytosis assay or the Cell Counting Kit-8 (CCK-8) and colony formation assay. The relationship between SOCS1 and let-7b-5p was confirmed with a dual-luciferase reporter.ResultsThe expression of cluster of differentiation 206 (CD206, a M2-like macrophage surface molecule) was significantly increased in M1 macrophages treated with let-7b-5p mimics, while CD206 expression was decreased in M-CMs treated with let-7b-5p inhibitors. Overexpression or knockdown of let-7b-5p significantly affected the expression of inflammatory factors in macrophages—including interleukin 10 (IL-10), IL-12, IL-13, and tumor necrosis factor alpha. Let-7b-5p downregulated the expression of suppressor of cytokine signaling 1 (SOCS1) and increased the phosphorylation of signal transducer and activator of transcription 1 (STAT1), STAT3, and STAT5a proteins in M-CMs and M1 macrophages with let-7b-5p mimics relative to the other groups. In addition, with the elevated expression of let-7b-5p, the phagocytosis by macrophages showed a commensurate and significant decrease. As a result, M-CMs treated with let-7b-5p inhibitors reduced the proliferation of PC-3 PCa cells.ConclusionsCollectively, these data indicated that let-7b-5p may regulate M2 polarization through the SOCS1/STAT pathway and that reversal of M2 differentiation by let-7b-5p inhibitors enhanced macrophage phagocytosis, ultimately inhibiting the proliferation of PCa cells.