Fig. 7

The pro-oncogenic effect of exosomes derived from TEMo on BC cell progression is partly dependent on miR-181a. A The relative expression level of plasma-derived circulating miR-181a in breast ductal carcinoma patients compared to healthy controls. A higher level of miR-181a was detected in BC plasma samples as compared with healthy controls, and, importantly, its expression level was correlated positively with tumor aggressiveness, as grade III tumors showed the highest expression of miR-181a with respect to grade I and II tumors. B, C Differential expression of miR-181 family members in TEMo and NEMo (B) as well as their corresponding exosomes (C). RT-qPCR results revealed a higher level of miR-181a in TEMo and TEMo-Exo compared to their normal counterparts. D The mean normalized ratio for miR-181a levels was assessed by RT-qPCR in MDA-MB-231 BC cells at 12 and 24 h time points. MDA-MB-231 BC cells were pre-treated with RNA polymerase inhibitor α-amanitin for 8 h before incubation with 100 μg/mL TEMo-Exo. The cells incubated with PBS and α-amanitin were used as a control. RT-qPCR results revealed that TEMo-secreted exosomal miR-181a is transferred to MDA-MB-231 BC cells in a time-dependent manner. E, F Representative flow cytometry histograms (E) and bar graphs (F) of cell cycle distribution in MDA-MB-231 BC cells in different conditions after 48 h of incubation. Flow cytometry results indicated transfection of miR-181a mimic caused BC cell progression similar to the cell cycle distribution observed in TEMo-Exo-treated BC cells. Additionally, inhibition of miR-181a resulted in the reduced distribution in the S and G2/M phases but induced an increased accumulation of cells in the sub G1 phase. Importantly, the TEMo-Exo-induced increases in S and G2/M proportions were partly prevented by reintroducing the miR-181a inhibitor, indicating that the promoting effect of TEMo-Exo on BC cell progression depends on miR-181a. G, H Representative photomicrographs (G) and bar graphs (H) illustrating the migration potential of MDA-MB-231 BC cells in different conditions after 24 h of incubation, assessed using a transwell assay. Overexpression of miR-181a recapitulated the promoting effect of TEMo-Exo on BC cell migration. However, miR-181a inhibition dramatically suppressed the phenotypes induced by TEMo-Exo in BC cells. Columns, mean of three different experiments; bars, SD. **P-value < 0.01, ***P-value < 0.001