Figure 4. Seed-dependent repression of miRNA targets by exosome-delivered miR-155 and miR-146a.

(a) Schematic for mimic experiment. (b,c) Relative mRNA levels of the miR-155 targets SHIP1 and BACH1 were measured via qRT–PCR in recipient cells that received exosomes with no mimics (No) (n=7), miR-155 seed mutant mimics (Seed) (n=4), with scrambled mimics (Scram) (n=3), or with miR-155-mimics (Mimic) (n=7). (d,e) qRT–PCR was preformed to assay the mRNA levels of the miR-146a targets, IRAK1 and TRAF6, following treatment with exosomes containing miR-146a mimics and controls as in b,c. Results are reported normalized to exosomes with no mimics added, which is set as 1. (f) Protein levels of TRAF6, IRAK1 and β-actin were determined via western blotting using lysates from miR-146a−/− BMDCs that received exosomes containing no mimics, seed mutant mimics or Wt mimics. Numbers below the blot represent relative protein levels with no mimics set as 1 following normalization to β-actin. (g) Schematic for 3′-UTR luciferase reporter assays in h,i. (h) Results from 3′-UTR luciferase reporter assays where miR-155−/− BMDCs were transfected with a pmiReport control vector, a BACH1 3′-UTR vector (BACH1), a BACH1 miR-155-binding site (bs) mutant vector (BACH1 mutant), or a 2mer-positive control vector. Transfected BMDCs were treated 6 h later with or without Wt exosomes and per cent change in luciferase activity of exosome treated BMDCs compared with no exosome treatment was calculated after 24 h (n=4). (i) Results from 3′-UTR luciferase reporter assay where miR-146a−/− BMDCs were transfected with a pmiReport control vector, a TRAF6 3′-UTR vector (TRAF6) or TRAF6 miR-146a bs mutant vector (TRAF6 mutant). Six hours later, the BMDCs were treated with or without Wt exosomes and per cent repression of luciferase activity was calculated 24 h after exosome transfer (n=4). Results represent two independent experiments. All data are presented as the mean±s.d. (error bars). *P<0.05; **P<0.01, ****P<0.0001; Student's t-test.