Induction Of M2 Polarization Of Macrophages By Lung Cancer-derived MicroRNAs

Background Macrophages are important cellular components of the tumor microenvironment and can be differentiated into M1 and M2 types.M1-type macrophages produce proinflammatory cytokines that kill microbes and tumor cells,whereas M2-type macrophages inhibit inflammatory signals and suppress anti-tumor immune responses in the tumor microenvironment,promoting tumor progression.In the tumor microenvironment,the polarization of macrophages is influenced by several factors,among which tumor cells and other stromal cells secrete exosomes and their components that regulate the differentiation,activation and function of macrophages and other immune cells.Reports have shown that tumor-derived exosomal mi RNAs not only have a role in promoting angiogenesis,tumor growth and metastasis,but also promote the conversion of macrophages into tumor-associated macrophages(TAM),which display an M2 phenotype and other immunomodulatory effects.Objective Two novel exosomal micro RNAs(tentatively designated as miR-A and miR-B)were found to be increasingly expressed in the plasma of lung cancer patients and had diagnostic values in our previous study(being published elsewhere).In this study,we focus on roles and mechanisms of miR-A and miR-B in the macrophage polarization.Methods 1)RNAs were extracted from cancer and adjacent tissues from lung adenocarcinoma patients,and culture media of lung adenocarcinoma cell lines for RT-qPCR analysis of expression of miR-A and miR-B;2)THP-1,a human monocyte/macrophage cell line,was stimulated with 200ng/mL of PMA overnight to transform the cells into M0 type,and then transfected with different concentrations of miR-A or miR-B.The cells were collected at different time points for qPCR detection of signature molecules of M1 and M2 phenotypes to determine the optimal action conditions;3)Under optimal experimental conditions,miR-A and miR-B were transfected into THP-1 cells and signature molecules for M1 and M2 phenotypes and proportion of M2 cells were detected by qPCR,ELISA,flow cytometry and immunofluorescence staining to find out if miR-A and miR-B promote M2 polarization;4)Several candidate target genes of miR-A and miR-B were detected by RT-qPCR and Western bolt to find out potential genes targeted by both miR-A and miR-B;5)Signature molecules for M1 and M2 phenotypes and proportion of M2 cells in the cells with the potential target gene knock-down by siRNA were detected by qPCR,ELISA,flow cytometry and immunofluorescence staining to observe if knock-down of the target genes have similar effects as transfection of miR-A or miR-B;6)The expression of CD68(total macrophages)and CD206(M2 macrophages)in lung cancer tissues of patients with high or low plasma levels of miR-A and miR-B were detected by immunofluorescence to observe the relationship of M2 polarization with expression of miR-A and miR-B.Randomly selected 5 sight fields under fluorescence microscopy were used for calculation of ratios of M2 macrophages.Results 1)miR-A and miR-B were increased expressed in lung cancer cells,and detected in the exosomes isolated from the culture media of cancer cells;2)The optimal conditions for observation of miR-A/B’s roles were 100 pmol for the transfection dose and 18h after transfection for signature molecules such as TNF-α,IL-1β and IL-10.3)Under the optimal conditions,transfection of miR-A or miR-B resulted in a significant increase in the expression of TNF-α(one of M1 markers)and an obvious decrease in the expression of IL-10 and other M2 markers such as CD206,AGS1,CCL18 and CCL22.The M2 polarization rates were 67.8±2.4% for miR-A and 59.75±5.3% for miR-B,significantly higher than 19.7±3.6% in the control THP-1 cells;(4)qPCR detection of x candidate target genes showed that LMF1 was a potential gene targeted by both miR-A and miR-B.5)After THP-1 activation by PMA and knockdown of LMF1 by siRNA,THP-1 tended to differentiate towards M2 type,with decreased expression of TNF-α,IL-1β,and increased expression of IL-10,CD206.The M2 polarization rate was 84.2±5.7% for LMF1-knockeddown THP-1 cells,compared with 14.5±4.8% in the control cells.knock-down of LMF1 induced effects similar to transfection of miR-A or miR-B indicate that LMF1 is a common target gene shared by both miR-A and miR-B.6)Immunofluorescence staining of the lung cancer tissues from the patients with high plasma levels of miR-A and miR-B showed that the M2 polarization rate 35.7±4.1%,compared with8.2±1.9% of lung cancer patients with low plasma levels of miR-A and miR-B(p less than0.001).These results indicated that the M2 polarization rate was positively correlated with the plasma levels of miR-A and miR-B.Conclusion Lung cancer-derived 2 novel exosomal micro RNAs,miR-A and miR-B,promote M2 polarization of macrophages probably by targeting LMF1.