Fig. 4

Exosomal transfer of miR-20a-5p from CAFs causes enhanced CRC cell proliferation and migration. A The mean normalized ratio for miR-20a-5p levels was assessed by RT-qPCR in SW480 CRC cells at 12 and 24-h time points. SW480 CRC cells were pre-treated with RNA polymerase inhibitor α-amanitin for 8 h before incubation with 100 μg/ml CAF-Exo. Inhibiting the endogenous source of miR-20a-5p using α-amanitin did not result in a significant change in the fold change of miR-20a-5p, suggesting the exosomal origin for this miRNA. The cells incubated with PBS and α-amanitin were used as a control. To confirm that this effect was solely attributed to exosomes and not influenced by any extraneous factors, we inhibited exosome secretion by introducing GW4869 in CAFs. RT-qPCR results revealed that CAF-secreted exosomal miR-20a is transferred to SW480 CRC cells in a time-dependent manner. B Cell proliferation exhibited a dose-dependent response, increasing as SW480 CRC cells were treated with increasing concentrations of CAF-derived exosomes. The highest level of cell proliferation was observed when the cells were treated with 100 µg/ml of CAF-derived exosomes. C Incubation of SW480 CRC cells with 100 μg/ml CAF-derived exosomes led to a considerable increase in the cell count, when compared to the negative control group. Importantly, transfection of miR-20a-5p inhibitor effectively diminished the promoting effect of CAF-derived exosomes on SW480 CRC cells. D Representative photomicrographs of the migration potential of SW480 CRC cells in different conditions after 48 h of incubation, assessed using the transwell assay. E Quantitative assessment of migrated cells revealed that the incubation of CRC cells with 100 μg/ml CAF-derived exosomes caused a significant increase in the migration rate of CRC cells. Of significant importance, the promoting effect of CAF-derived exosomes on SW480 CRC cell migration was partially diminished when SW480 CRC cells were concurrently transfected with the miR-20a-5p inhibitor. F-I The mean normalized ratios measured for proliferation markers (i.e., c-Myc and CDKN1A) and migration markers (N-cadherin and MMP2) indicated that incubation of SW480 CRC cells with 100 μg/ml CAF-derived exosomes led to an up-regulation in the expression levels of these markers. However, no significant changes were detected when SW480 cell were incubated with NF-derived exosomes. Notably, transfection of miR-20a-5p inhibitor into CAF Exo-treated SW480 CRC cells reduced the promoting effects of CAF-derived exosomes on the expression levels of these proliferation and migration markers. Columns represent means of three different experiments; bars represent SD. *P < 0.05, **P < 0.01, ***P < 0.001