Effect Of MiR-146a On Tumor-associated Macrophage Differentiation And Its Mechanism

Macrophages at least can be divided into two types:M1type and M2type, which are classically activated macrophages and alternatively activated macrophages. M1macrophages have very strong sterilization and antigen-presenting capacity, and M2macrophages have function on repair the damage, promote angiogenesis and tissue remodeling. Macrophage infiltrating into tumor mass is called tumor associated macrophage (TAM). TAM is a kind of highly heterogeneous populations of cells, typing between M1to M2. In recent years, gradually increased evidence showed that TAM could promote tumor development, including promoting tumor growth, invasion and metastasis. At present, the M1and M2ratio are used to measure the ability of macrophages in inhibiting tumor cells in tumor mass.The study was based on previous microarray results of our lab, which tested expression changes of microRNAs during TAM differentiation in the model of mouse breast cancer, and miR-146a was the most obviously downregulatd one in the chip result. It was reported that miR-146a could negatively feedback to regulate of LPS-induced expression of inflammatory factors, and our previous experiment results also confirmed this view. Intreguingly, as a classical inhibitory factor of inflammation, the expression of miR-146a was unexpectedly down-regulated in TAM. This contradiction suggested miR-146a might be regulated in some special way in TAM, and even hinted that miR-146a might have a more complex function in the immune regulation.This research verified the miRNA microarray results on mouse4T1transplanted tumor model to, and also confirmed a downward trend of miR-146a in TAMs from human breast cancer, gastric cancer, and colonic carcinoma, which was consistent with results obtained from mouse.To understand the reason that expression of miR-146a was down-regulated in TAM, we stimulated mouse peritoneal macrophages (PEC) with the cytokines in tumor microenvironment and cell culture supernatant, but none of them could induce down-regulation of miR-146a expression in macrophages. Then, we found NF-κB p50subunit was elevated in TAMs in clinical specimens from breast cancer patients. By the experiment of ChIP and overexpression of P50,we proved p50was capable to bind to miR-146a promoter, and involved in the negative regulation of miR-146a.In order to further understanding of the miR-146a features in TAM, the synthesized miR-146a mimics and inhibitor were transfected into PEC, and then LPS and IL-4were used to chanllenged them respectively. We Found that inhibition of miR-146a in PEC could promote LPS-induced the expression of IL-1b, IL-6and iNOS, and inhibit expression of IL-4-induced IL-10, Argland PDGF as markers of M2macrophages, though some of the M2macrophage markers such as YM1, Fizz and CCL17did not be affected. It was also showed in vivo that co-injected with miR-146a antagmir-transfected PEC, growth of4T1cells were more slowly than with NC-transfected PEC, suggesting that downregulation of miR-146a in TAM could suppress the growth of tumor and played a negatively regulatory role in tumor growth.In conclusion, this study found that, miR-146a significantly reduced in TAM from human and mouse breast cancer. Its down-regulation might be caused by increased expression of NF-κB p50subunit in TAM. Inhibition of miR-146a expression could promote macrophage M1polarization and partial inhibit of M2polarization, and also suppressed tumor growth. It suggested that miR-146a in TAM played a role of negative feedback regulation on tumor growth.This paper provided new data, molecular mechanisms and new method on tumor treatment.