Inhibition of let-7b-5p contributes to anti-tumorigenic macrophage phenotype through SOCS1/STAT pathway in prostate cancer

Abstract BackgroundDysfunction of microRNAs (miRNAs) is a major cause of aberrant expression of inflammatory cytokines and contributes to macrophage polarization. Previous studies have shown that miRNA let-7 is associated with cell differentiation, and the expression of let-7b-5p is significantly increased 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 the blood monocytes from healthy male donors. M1 macrophages were polarized by stimulating overnight with 100 ng/ml lipopolysaccharides and 100 ng/ml IFN-γ, while conditioned medium from PC-3 cells were used to induce prostatic macrophages (M-CMs) in vitro, and then let-7b-5p mimics or inhibitors were transfected into M1 and M-CMs for 72 hours respectively. The expression of cluster of differentiation 206 (CD206) in each group was detected by a High Throughput Connotation of Imaging System. Quantitative real-time polymerase chain reaction (qRT-PCR) was executed to examine the expression of inflammatory cytokines IL-10, IL-12, IL-13, TNF-alpha and let-7b in macrophages. SOCS1 protein level was evaluated by ELISA, and the phosphorylation difference of STAT family member proteins was analysized by CST signal pathway chip. Phagocytosis of macrophages and the effect of macrophages on the proliferation of prostate cancer cell PC-3 were evaluated by phagocytosis assay or Cell Counting Kit-8 (CCK-8). The relationship between SOCS1 and let-7b-5p was confirmed with the dual-luciferase reporter.ResultsThe expression of cluster of differentiation 206 (CD206), an 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 compared with the other groups. In addition, with high expression of let-7b-5p, the phagocytosis of macrophages showed a significant decrease. As a result, M-CMs treated with let-7b-5p inhibitors reduced the proliferation of PC-3 PCa cells.ConclusionsCollectively, these data indicate that let-7b-5p may regulate M2 polarization through the SOCS1/STAT pathway, and reversal of M2 differentiation by let-7b-5p inhibitors can enhance macrophage phagocytosis, ultimately inhibiting the proliferation of PCa cells.Trial registrationNot applicable