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. 2019 May 17;17:49–62. doi: 10.1016/j.omtn.2019.05.002

Figure 3.

Figure 3

Tie2 Is Targeted by miR-15a and -16 through an Unusual Binding Site on Tie2 mRNA, and It Is Involved in the Proangiogenic Effect of miR-15a and -16 Inhibition in HUVECs In Vitro

(A) Luciferase activity at 48 h post-co-transfection of HEK293T cells with pre-miR-15a (miR-15a), pre-miR-16 (miR-16), a combination of the two (miR-15a/16), or miR-scrambled oligonucleotides (Scr) and plasmid containing luciferase open reading frame (pLUC), followed by a portion of 50 nt of the TEK-coding sequence surrounding the putative miRNA-binding site (pLUC-TEK). A mutated version of the miRNA-binding site was used as a control (pLUC-TEKmut). A schematic of the plasmid construct is also provided. *p < 0.05, ***p < 0.001 versus pLUC-TEK control Scr; †p < 0.05, ††p < 0.01 versus pLUC-TEK to the pre-miRNA matching condition (n = 5). (B) Human umbilical vein endothelial cells (HUVECs) were transfected with a Scramble oligo (Scr), pre-miR-15a (miR-15a), or pre-miR-16 (miR-16) (5 nM) to increase their respective mature miRNA expression. miR-15a and -16 expressions were assessed by RT-PCR and normalized to small nuclear U6 expression. Tie2 mRNA (C) or protein (D) expressions were assessed by RT-PCR and western blot and normalized to 18S and β-actin, respectively. n = 3 for each condition. (E) HUVECs were transfected with a Scramble oligo (Scr) or an anti-miRNA against miR-15a, miR-16, or the two anti-miRNAs simultaneously (total concentration of 50 nM) to inhibit their activity. Tie2 protein expression was assessed by western blot and normalized to β-actin. n = 9 for each condition. *p < 0.05 versus Scr condition. (F) Effect of inhibition of Tie2 on miR-15a/16 inhibition-induced angiogenesis was assessed in HUVECs transfected with a Scramble oligo (Scr) or an anti-miRNA against miR-15a or miR-16 (50 nM) and treated with Tie2 inhibitor (Tie2-I, 5 μM) or DMSO control. The angiogenic capacity was measured by network formation on Matrigel, calculated as total length in millimeters and expressed as a percentage of the Scr and DMSO conditions. Representative pictures are provided (scale bar, 200 μm). n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. All results are expressed as mean ± SEM.