Different expression of miRNA between the exosomes secreted by monocyte and M2 macrophage

The macrophages in tomor microenvironment in were alike M2 macrophages which contribute to tumor progression and migration. Since macrophages can secret lots of exosomes and M2 macrophage can induce the imbalance of Treg/Th17 ratio in EOC tumor environment, we want to investage the different expression of miRNA between the exosomes secreted by monocyte and M2 macrophage. Thus to see if the microRNA in exosomes secreted from M2 macrophage paly a big role in the T cell imbalance. Thp-1 cell was induced into M2 macrophage, and exosomes was collected. The Exosome from untreated Thp-1 cells were used as control. The M2 macrophages were induced by PMA (50 ng/ml) and IL-4 (20 ng/ml) for 24h. Then the culture medium was change into RPMI1640 and culture for 24h. In order to isolate exosomes, we centrifuge the supernatants for two times (1000g × 10min, 3000g × 30min to deplete the cell or fragments), and then add the total exosome isolation kit (Life technology) over night, and then centrifuge for 10000g×1h. Total RNA was extracted by mirVanaTM RNA Isolation Kit (Applied Biosystem p/n AM1556)