EFFECTS OF HYPOXIC HEPATOMA-DERIVED EXOSOME miR-1260b ON M2 MACROPHAGES AND MECHANISM

Abstract

Objective To explore the effects of the hypoxic hepatoma-derived exosome miR-1260b on the M2 subtype of tumor-associated macrophages and the underlying mechanism. Methods MHCC97H (97H) cells were treated with 100 μmol/L CoCl2 for 24 h to obtain hypoxic 97H cells. THP-1 cells were treated with propylene glycol methyl ether acetate (PMA) for 24 h to derive M0 macrophages. The relative expression of CD11b in THP-1 cells was measured by real-time quantitative PCR (RT-qPCR). After co-culture with normoxic/hypoxic 97H cells for 48 h, the relative gene expression of CD163, CD206, and TNF-α in M0 macrophages was measured by RT-qPCR. The exosomes of normoxic/hypoxic 97H cells were extracted by low temperature differential centrifugation. A transmission electron microscope was used to observe the shape of the exosomes. The particle diameter was measured using a nanoparticle tracking analyzer. Western blot was used to determine the relative expression of apoptosis-inducing factor 6-interacting protein (Alix) and calnexin in the exosomes. After co-culture with the exosomes for 48 h, the relative expression of CD206 in M0 macrophages was measured by RT-qPCR. The exosomes labeled with Dil stain were co-cultured with M0 macrophages for 24 h, and then M0 macrophages were examined for the fluorescence reaction using a fluorescence microscope. The expression of miR-1260b in the exosomes was measured by RT-qPCR. After M0 and M2 macrophages were transfected with miR-1260b Mimic/Inhibitor, the expression of CD206 and TNF-α in the cells was determined by RT-qPCR. After co-culture with normoxic/hypoxic 97H cells transfected with miR-1260b Inhibitor, M0 macrophages were measured for the relative expression of CD206 and TNF-α by RT-qPCR. Results There was significantly high expression of CD11b in M0 macrophages derived from THP-1 cells after 24 h PMA treatment (t=78.14,P<0.05). M0 macrophages co-cultured with hypoxic 97H cells showed significantly increased expression of CD163 and CD206 and significantly decreased expression of TNF-α (t=14.23-46.88,P<0.05). The exosome particles secreted by hypoxic 97H cells were successfully isolated and extracted, which could be phagocytized by M0 macrophages. Western blot showed that the exosome particles had high expression of Alix protein and very little expression of calnexin. The results of RT-qPCR showed that co-culture with hypoxic hepatoma-derived exosomes significantly increased the expression of CD206 in M0 macrophages (t=17.06,P<0.05). The expression of miR-1260b was significantly increased in hypoxic hepatoma-derived exosomes (t=12.09,P<0.05). M0 macrophages highly expressed CD206 and lowly expressed TNF-α after transfection with miR-1260b Mimic (t=8.82,8.22,P<0.05), and highly expressed TNF-α after transfection with the Inhibitor (t=16.88,P<0.05). Pre-transfection of miR-1260b Mimic into normoxic 97H cells significantly increased the expression of CD206 and significantly decreased the expression of TNF-α (t=26.95,21.45,P<0.05). Pre-transfection of miR-1260b Inhibitor into hypoxic 97H cells significantly changed the expression of CD206 and TNF-α (t=9.09,12.54,P<0.05). Conclusion Hypoxic hepatoma-derived exosomes can induce M2 macrophage polarization. miR-1260b is a key target for macrophage polarization by hypoxic hepatoma cell-derived exosomes, which promotes the immune escape of hepatoma cells.

Keywords

• liver neoplasms|cell hypoxia|exosomes|micrornas|transfection|macrophages|tumor escape