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. 2016 Mar 30;7(21):30420–30439. doi: 10.18632/oncotarget.8483

Figure 3. MiR-21, shuttled by exosomes, modulates RhoB expression in HUVECs.

Figure 3

(A) Real time PCR analysis showed that RhoB mRNA expression decreased in HUVECs treated with Curcu-exosomes compared to control exosomes. Expression of RhoB was evaluated in HUVECs transfected with 2-Ome-miR-21 (miR-21 inhibitor) treated or not with 20 μg/ml of control exosomes (miR-21 inhibitor + Exo 20 μg/ml) and Curcu-exosomes (miR-21 inhibitor + Curcu-Exo 20 μg/ml). Expression of RhoB was also evaluated in HUVECs transfected with miR-21 mimic (miR-21 mimic) treated or not with 20 μg/ml of control exosomes (miR-21 mimic + Exo 20 μg/ml) and Curcu-exosomes (miR-21 mimic + Curcu-Exo 20 μg/ml). Values (FOI: fold of induction) are the mean ± SD of 3 independent experiments *p ≤ 0.05, **p ≤ 0.01. (B) Histogram shows the MFI (Mean Fluorescence Intensity) relative to the expression of RhoB in HUVECs after treatment with low serum medium (Control), 20 μg/ml of exosomes (Exo 20 μg/ml) and 20 μg/ml of Curcu-exosomes (Curcu-exo 20 μg/ml). Expression of RhoB was evaluated, with FACS analysis, in HUVECs transfected with 2-Ome-miR-21(miR-21 inhibitor) and treated with 20 μg/ml of control exosomes (miR-21 inhibitor + Exo 20 μg/ml) and Curcu-exosomes (miR-21 inhibitor + Curcu-Exo 20 μg/ml). Surface expression of VCAM1 was evaluated in HUVECs transfected with miR-21 mimic (miR-21 mimic) and treated with 20 μg/ml of control exosomes (miR-21 mimic + Exo 20 μg/ml) and 20 μg/ml of Curcu-exosomes (miR-21 mimic + Curcu-Exo 20 μg/ml). Values are the mean ± SD of 3 independent experiments *p ≤ 0.05 **p ≤ 0.01. (C) Curcu-exosomes inhibit HUVECs migration. Addition of control exosomes (20, 50 μg/ml) to the upper wells of the chamber induces dose-dependent increase of HUVEC migration, the addition of Curcu-exosomes reverts this effects. Values are the mean ± SD of 3 fields in three independent experiments *p ≤ 0.05, **p ≤ 0.01. The ability of migrating of HUVECs transfected with 2-Ome-miR-21 (miR-21 inhibitor) treated or not with 20 μg/ml of control exosomes (miR-21 inhibitor + Exo 20 μg/ml) and with 20 μg/ml of Curcu-exosomes (miR-21 inhibitor + Curcu-Exo 20 μg/ml), was evaluated. The ability of migration of HUVECs transfected with miR-21 mimic (miR-21 mimic) treated or not with 20 μg/ml of control exosomes (miR-21 mimic + Exo 20 μg/ml) and with 20 μg/ml of Curcu-exosomes (miR-21 mimic + Curcu-Exo 20 μg/ml) was also measured.

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