Transfer of miR-135b derived from HR-MM cells to HUVECs via exosomes. HUVECs/β-gal (HUVECs transfected with pMIR-reporter β-gal control vector) were cultured with PKH67-labeled exosomes derived from the parental cells (RPMI8226) transfected with Cy3-pre-miR-135b. The Cy3-miR-135b signals were detected in (A) the cytoplasm of HUVECs (red), and (B) green signals indicate PKH67-labeled exosomes. (C) Cy3-miR-135b signals are colocalized with PKH67 in HUVECs (yellow). Parts of areas in (A-C) are enlarged in (D-F), respectively. Nuclear counterstaining was performed using 4′,6-diamidino-2-phenylindole (DAPI) (blue). The scale bar indicates 10 µm. (G) Luciferase reporter vector for assessing miR-135b–specific activity contained complementary miR-135b sequences in its 3′-UTR. The normalized firefly luciferase activity was obtained by firefly luciferase activity/β-gal activity. Sensor vector: luciferase activity of HUVECs/Luc/β-gal (HUVECs cotransfected with luciferase reporter vector and β-gal control vector) treated with RPMI8226-HR1%O2-exosomes was significantly reduced compared with control (HUVECs/Luc/β-gal without exosomes) (**P < .01, n = 3). RPMI8226-HR/anti-miR135b exosomes (miR-135b–deactivated exosomes of RPMI8226-HR cells) could not reduce the luciferase activity in HUVECs (RPMI8226-HR/anti–miR135b-exo vs RPMI8226-HR/con-exo: #P < .01, Student t test, n = 3). In the mutated sensor vector, there was no difference in luciferase activity with or without exosomes. (H-I) Tube formation assay of HUVECs cultured under 1% O2 with RPMI8226-HR1%O2/control exosomes (H) and with RPMI8226-HR1%O2/anti-miR135b exosomes (I). The scale bar indicates 500 µm. (J) The tubelike structures determined by pixel density are reduced by the addition of RPMI8226-HR1%O2/anti-miR135b exosomes compared with control (*P < .05 vs control, Student t test, n = 3). Values are means ± SD of 3 independent experiments, with each performed on a different day. exo, exosome; con, control.
