The Study Of HA Induced Formation Of M2-like Tumor-associated Macrophages Via Regulating MiRNA

Objective:Our study aims to investigate the role of hyaluronan(HA)in the immune regulation of tumor:the expression of circulating M2-like monocytes in patients with breast cancer and the relationship between circulating M2-like monocytes and plasma HA,especially the underlying mechanisms of the role of HA in promoting the polarization of non-activated macrophages to a M2-like TAMs phenotype.Methods:1.The expression of circulating CD14~+CD163~+/CD14~+CD204~+/CD14~+CD163~+CD204~+M2-like monocytes in patients with breast cancer and benign disease and healthy controls were determined by flow cytometry,and the relationships of M2-like monocytes and clinical parameters of breast cancer were assessed.2.The expression of M2-like TAMs and HA was determined in pathological tissues by immunohistochemistry.3.The concentrations of plasma HA,CEA,and CA15–3 were measured by chemiluminescence method,and the correlation between plasma HA and circulating M2-like monocytes was analyzed.4.Peripheral blood monocytes from healthy donors were isolated from frozen PBMCs by CD14 Micro-Bead-mediated sorting system.Monocytes were incubated with different concentrations and molecular weights of hyaluronan for 72 h.5.The expression of M2-related cytokines or mRNA(Human CCL18,CCL22,IL-10,CCR2 and so on)in the culture supernatants and monocytes were measured by ELISA and RT-qPCR,respectively.6.RNA from human primary macrophages treated with or without HA for 72 h and miRNA microarray analysis was performed and used to compare the miRNA expression profiles.7.The online software including TargetScan,miRDB,and miRWalk were used to analyzed the potential targets.8.To confirm the roles of miRNA in macrophage polarization,we transfected THP1-derived macrophages with miRNA mimics for overexpression of miRNA and inhibitors for inhibition of miRNA expression.9.The potential target of miRNA was testified by RT-qPCR and western blot.10.Collecting the plasma and monocytes of patients with solid tumors,and the levels of plasma HA were detected by chemiluminescence method.Expression of miRNA,CCL18,IL-10,and the potential target of miRNA in monocytes of peripheral blood of solid tumors were determined by RT-qPCR.The associations between these markers were analyzed.Results:1.Our results showed that the percentages of circulating M2-like monocytes especially CD14~+CD204~+monocytes from breast cancer patients were significantly higher than that from breast benign disease and healthy controls.2.In contrast to CEA and CA15-3,circulating CD14~+CD204~+monocytes possessed higher sensitivity and specificity.3.We found that CD204~+M2-like macrophages and HA were accumulated in the stroma of breast cancer,which were higher than that in benign tissue.4.The sensitivity and specificity of the combination of circulating CD14~+CD204~+M2-like monocytes and HA were significantly higher than that of CEA and CA15-3.5.Higher frequency of circulating CD14~+CD204~+M2-like monocytes were significantly associated with TNM stage,histological differentiation,and lymph node metastasis.6.We found that the M2-like polarization of macrophages induced by 50μg/mL INT-HA fragments was particularly evident,whereas the effects of HMW-HA and LMW-HA were not obvious.7.Our study found that M2-like macrophages polarization was mainly induced by INT-HA via TLR4.8.We compared miRNA expression in the untreated cells versus INT-HA treated monocytes by miRNA microarray analysis,and RT–qPCR validated the dramatic decrease in miR-935 expression upon INT-HA-induced differentiation of monocytes into macrophages.9.Down-regulation of miR935 in macrophages is HA-dependent and TLR4-specific.10.MiR-935 may play an important role in M2 macrophage polarization activated by INT-HA.11.miR-935 may induce macrophages to M2-like differentiation through it’s target gene C/EBPβ.Conclusion:1.The frequency of circulating CD14~+CD204~+M2-like monocytes was positively correlated to plasma HA levels.The combination of circulating CD14~+CD204~+M2-like monocytes and plasma HA could be identified as the combined biomarkers for the diagnosis of breast cancer.;2.INT-HA could modulates the polarization of macrophages to an M2-like phenotype;3.Our study demonstrates that INT-HA modulates the polarization of macrophages to an M2-like phenotype through TLR4-miR-935-C/EBPβpathway,which may be used as a novel target for the therapy of breast cancer.