MiR-19a-3p Suppresses Breast Cancer Metastasis By Regulating The Phenotype Of TAMs Via Inhibiting Fra-1/IL-6/Stat3Signalling Pathway

One of the hallmarks of malignancy is the polarization of tumor-associated macrophages (TAMs) from a pro-immune (M1-like) phenotype to an immune-suppressive (M2-like) phenotype. However, the molecular basis of the process is still unclear. MicroRNA (miRNA) is comprised by a group of small, non-coding RNAs which are broadly expressed by a variety of organisms and involved in such cell behaviors as suppression or promotion of tumorigenesis. Based on these prvious findings, we tried to explore the relationship between the function of miRNA and TAMs in tumor progression, and how miRNAs regulate the phenotype of TAMs and the progress of tumor metastasis. Firstly, we used mouse primary macrophages, mouse leukemia macrophage cell line RAW264.7, human mononuclear macrophage cell ine U937as macrophage model, and human breast cancer cell line MDA-MB-231, mouse breast cancer cell line4T1were used as breast cancer cell model. We tested how the tumor microenvironment regulates the miRNA expression of TAMs. Based on the function of Fra-1in regulating the polarization of TAMs, we predicted the potential targeting miRNAs of Fra-1with Target Scan programme. Here, we demonstrate that miR-19a-3p, broadly conserved among vertebrates, has potential binding site in3’UTR of Fra-1and is down-regulated in RAW264.7and U937macrophage cells of M2phenotype by conditioned medium of4T1and MDA-MB-231breast tumor cells:This down-regulation correlated with an increased expression of the Fra-1gene which was reported to play the role of a pro-oncogene by supporting invasion and progression of breast tumors. We found significant up-regulation of miR-19a-3p in RAW264.7macrophages, following the transfection with a synthesized miR-19a-3p mimic, which leads to significant suppression in expression of Fra-1gene. Additionally, we confirmed that miR-19a-3p bind to site the3’UTR of Fra-1with a psiCHECK luciferase reporter system. Furthermore, transfection of RAW macrophage cells with the miR-19a-3p mimic decrease expression of VEGF, STAT3and p-STAT3, which are downstream gene of Fra-1signaling. By real-time PCR, we found that miR-19a-3p down-regulates the M2markers of macrophages as well as suppressing the M2cytokines, such as IL-4, IL-6and IL10.Based on the in vitro results, we synthesized miR-19a-3p agomir and applied it to animal experiment by injecting it intratumor to overexpress miR-19a-3p in TAMs in vivo. After6times of injection, we removed the primary tumor to analysis the phenotype of TAMs in tumor tissue by IHC, IF and flow cytometry. We found that miR-19a-3p down-regulated the proportion of M2macrophages within tumor. Most importantly, by HE staining, we found that the number and size of metastasis nudes were down-regulated by miR-19a-3p, indicating that the metastatic capacity of4T1breast tumor cells on lung tissues was significantly impaired. However, the growth rate of primary tumor was not affected. Taken together, these findings indicated that miR-19a-3p is capable of inhibiting the M2phenotype of TAMs and the low expression of this microRNA plays an important role in up-regulating the expression of Fra-1and induction the polarization of M2macrophage. Therefore, combined with nanoparticle-mediated delivery approach, it is possible that miR-19a-3p could also become a potentially useful therapeutic target for the treatment of breast cancer.