FIGURE 4.

Exosomal angiogenic miRNAs from cancer cells regulate angiogenesis in endothelial cells. A, 4T1-siLuc cells were treated with 10 μm GW4869 at the start of the co-culture for a total of 48 h (p < 0.001). Each error bar is presented as the mean ± S.E. (n = 3). **, p < 0.005, as compared with control. B, heat map showing expression levels of the exosomal miRNAs isolated from HEK293, MCF10A, MCF7, and MDA-MB-231. Blue to red, color range gradient of mean abundance. C, the expression level of miR-210 in exosome isolated from MCF10A, MCF7, MDA-MB-231-D3H1 (MM231-D3H1), or MDA-MB-231-D3H2LN (MM231-D3H2) cells. Each error bar is presented as the mean ± S.E. (n = 3). **, p < 0.005, as compared with MCF10A. D, expression of exosomal miR-210 in exosomes isolated from parental 4T1 cells or 4T1-nSMase2-KD cells. Each error bar is presented as the mean ± S.E. (n = 4). **, p < 0.005, as compared with 4T1-control cells. E, HUVECs were co-cultured with parental 4T1 cells or 4T1-nSMase2-KD cells for 48 h. RNA was isolated from the HUVECs at 48 h after the start of co-culture, and the expression of exosomal miR-210 in the HUVECs was analyzed by qRT-PCR. Each error bar is presented as the mean ± S.E. (n = 3). *, p < 0.05, as compared with 4T1 control cells. F, parental 4T1 cells or 4T1-nSMase2-KD cells were co-cultured with HUVECs for 48 h, and the expression levels of ephrin-A3 (target of miR-210) were analyzed by qRT-PCR. Each error bar is presented as the mean ± S.E. (n = 3). *, p < 0.05, as compared with 4T1-control cells.