Fig. 3

ExomiR-155 mediates the adipocyte metabolism by downregulating PPARγ. The adipocytes in 50 μg of exosomes purified from cancer-associated conditioned medium (CA-CM). (a) Exosomes originating from CA-CM viewed by electron microscopy (scale bar, 200 nm). (b) Exosomes from CA-CM were analysed by western blot. (c) NanoSight analysis of exosomes derived from CA-CM. (d) 4 T-1 were cocultivated in the presence or absence of adipocytes. After 3 days, exosomal miRNAs were further verified by qPCR. And RNA was extracted from the adipocytes and subjected to qPCR analysis with primers specific to mature miRNA. (e) The predicted miR-155 binding site in the 3’UTR of the PPARγ gene from TargetScan. (f) The GV272 vector containing the 3’UTR of the target gene harbouring wild-type (wt) or mutated (mt) miRNA binding sites was transfected into HEK 293 T cells stably expressing miRNA or empty vector (as a control). Luciferase activity was analysed at 48 h post-transfection, and the ratio of firefly luciferase activity to Renilla luciferase activity is shown. (g) Breast cancer cells were transfected with miRNA-155 pre-miRNA or miR-155 inhibitor, mature adipocytes were transfected with miR-155 mimic as the positive control and the control vector was applied as the negative control. Mature adipocytes cultured in the presence or absence of tumour exosomes for 3 days were stained with red oil, and Western blot analysis of related protein expression in different groups (h). Data are presented as the mean ± S.D. of at least three independent experiments. * P < 0.05 versus control values, # P < 0.05 versus control values as positive group