Figure 6.
Figure 6. Regulation of HGF/cMET signaling in endothelial cells by exosomal miR-340 derived from young BMSCs. (A) Representative Western blots of the expression of cMET and HGF in HUVECs after treatment with older BMSC exosomes transfected with miR-340 mimics (miR-340 exo.) or negative control miR (control miR exo.). (B) The hsa-miR-340-binding site spanning nucleotides 1487–1508 (accession no. NM_000245) of the human cMET 3′-UTR was predicted to be evolutionarily conserved. The seed sequence of hsa-miR-340 (AAAUAU) is shown in red. (C) Luciferase reporter assay using a reporter plasmid containing the cMET 3′-UTR with the miR-340-binding site. The luciferase reporter vector to assess miR-340-specific activity contained complementary miR-340 sequences in its 3′-UTR. Normalized firefly luciferase activity was obtained according to firefly luciferase activity/β-gal activity. Sensor vector: the luciferase activity of HUVECs/Luc/β-gal (HUVECs co-transfected with luciferase reporter and β-gal control vectors) treated with young BMSC exosomes was significantly reduced in comparison with control cells (HUVECs only, *P < .01, n = 3). miR-340 exosomes (older BMSC exosomes transfected with miR-340 mimics) also reduced the luciferase activity in HUVECs in comparison with control miR exosomes (older BMSC exosomes transfected with negative control miR) (#P < .01, Student t test, n = 3). Using the mutated sensor vector, there was no difference in luciferase activity with or without exosomes. (D) Schematic of the endothelial cell tube formation assay to confirm the antiangiogenic effect of miR-340 exosomes. HUVECs were cultured on Matrigel in 3 types of medium (basal medium, basal medium supplemented with conditioned medium from RPMI8226-HR cells, or basal medium supplemented with HGF). (E) Endothelial tube formation in response to older BMSC exosomes transfected with miR-340 mimics (miR-340 exo.) observed under a bright field by phase-contrast microscopy. Scale bar, 500 µm. (F) Quantification of tube formation as shown in panel E. The conditioned medium from HR-MM (RPMI8226-HR) cells and HGF induced endothelial tube formation in vitro. Induction of tube formation by conditioned medium of HR-MM cells and HGF was inhibited by older BMSC exosomes transfected with miR-340 mimics (miR-340 exosomes) in comparison with the control (older BMSC exosomes transfected with negative control miR; control miR exosomes) (*P < .01, Student t test). Values represent the mean ± SD. (G) Immunohistochemical staining of CD31 (red) and p-cMET (green) in Matrigel plugs seeded with RPMI8226-HR cells at 3 weeks after transplantation into nude mice. Scale bar, 200 µm. med., medium.

Regulation of HGF/cMET signaling in endothelial cells by exosomal miR-340 derived from young BMSCs. (A) Representative Western blots of the expression of cMET and HGF in HUVECs after treatment with older BMSC exosomes transfected with miR-340 mimics (miR-340 exo.) or negative control miR (control miR exo.). (B) The hsa-miR-340-binding site spanning nucleotides 1487–1508 (accession no. NM_000245) of the human cMET 3′-UTR was predicted to be evolutionarily conserved. The seed sequence of hsa-miR-340 (AAAUAU) is shown in red. (C) Luciferase reporter assay using a reporter plasmid containing the cMET 3′-UTR with the miR-340-binding site. The luciferase reporter vector to assess miR-340-specific activity contained complementary miR-340 sequences in its 3′-UTR. Normalized firefly luciferase activity was obtained according to firefly luciferase activity/β-gal activity. Sensor vector: the luciferase activity of HUVECs/Luc/β-gal (HUVECs co-transfected with luciferase reporter and β-gal control vectors) treated with young BMSC exosomes was significantly reduced in comparison with control cells (HUVECs only, *P < .01, n = 3). miR-340 exosomes (older BMSC exosomes transfected with miR-340 mimics) also reduced the luciferase activity in HUVECs in comparison with control miR exosomes (older BMSC exosomes transfected with negative control miR) (#P < .01, Student t test, n = 3). Using the mutated sensor vector, there was no difference in luciferase activity with or without exosomes. (D) Schematic of the endothelial cell tube formation assay to confirm the antiangiogenic effect of miR-340 exosomes. HUVECs were cultured on Matrigel in 3 types of medium (basal medium, basal medium supplemented with conditioned medium from RPMI8226-HR cells, or basal medium supplemented with HGF). (E) Endothelial tube formation in response to older BMSC exosomes transfected with miR-340 mimics (miR-340 exo.) observed under a bright field by phase-contrast microscopy. Scale bar, 500 µm. (F) Quantification of tube formation as shown in panel E. The conditioned medium from HR-MM (RPMI8226-HR) cells and HGF induced endothelial tube formation in vitro. Induction of tube formation by conditioned medium of HR-MM cells and HGF was inhibited by older BMSC exosomes transfected with miR-340 mimics (miR-340 exosomes) in comparison with the control (older BMSC exosomes transfected with negative control miR; control miR exosomes) (*P < .01, Student t test). Values represent the mean ± SD. (G) Immunohistochemical staining of CD31 (red) and p-cMET (green) in Matrigel plugs seeded with RPMI8226-HR cells at 3 weeks after transplantation into nude mice. Scale bar, 200 µm. med., medium.

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