Figure 4.

Incorporation of EXOmotifs enhances miRNA delivery and target inhibition.

a) Diagram illustrating the transwell experiment.

b) miRNA expression for endogenous basal miR-34c (grey bar), induced wild-type miR-34c over the basal (blue), miR-34c-CAUG (green) and miR-34c-CGGGAG (red) in the donor cells and expressed as the number of copies of each miRNA per 10³ copies of miR-103-3p as an endogenous control.

c) miRNA expression for basal miR-34c (grey bar), induced wild-type miR-34c over the basal (blue), miR-34c-CAUG (green) and miR-34c-CGGGAG (red) in the recipient cells after co-incubation with the donor cells overexpressing these miRNAs respectively, as indicated in the x-axis and normalized per 10³ copies of miR-103-3p.

d) Average number of copies of each miRNA induced in the donor cells above the levels measured in control Scr cells (second column) and the number of copies in the recipient cells over the levels in cells cocultured with the O/E-Scr donors (third column). Values are normalized per 10³ copies of miR-103-3p. The last column represents the ratio between the second and third columns.

e) Change in gene expression for miR-34c targets in the recipient cells induced by the coculture with miR-34c-WT, miR-34c-CAUG or miR-34c-CGGGAG donor cells as indicated in the x-axis and relative to recipient cells co-incubated with O/E-Scr donors. n=3; Data are expressed as mean ± SEM. *P≤0.05 motif-containing vs WT-miR-34c for each gene (Kruskal-Wallis followed by Mann-Whitney-U test).

f) Proposed model for the sEV/exosomal sorting and cellular retention of miRNAs mediated by the cell-specific repertoire of EXO- and CELLmotifs. In this, “reader” proteins (i.e., Alyref and Fus for motif CGGGAG) recognize the miRNA motifs that promote sorting of EXOmotif-harboring miRNAs into exosomes/multivesicular bodies (MVB) or cellular retention (CELLmotif). The released exosomes then deliver their miRNA cargo to nearby or distal cells leading to changes in gene expression and cellular function.