miR-1233-3p Inhibits Angiopoietin-1-Induced Endothelial Cell Survival, Migration, and Differentiation

Figure 1. Ang-1 regulates miR-1233-3p in HUVECs. (A,B) miR-1233-3p levels measured in HUVECs exposed for 2, 4, 6, 10, 24, 48, and 72 h to PBS or Ang-1 (300 ng/mL). (C) Expression of GOLGA8A mRNA in HUVECs exposed for 2, 4, 6, and 10 h to PBS or Ang-1. (D) Levels of miR-1233-3p in HUVEC exosomes after 24 h of PBS or Ang-1 (300 ng/mL) treatment. (E) Expression levels of miR-1233-3p in HUVECs treated with control or Tie-2 blocking antibodies and incubated with PBS or Ang-1 (300 ng/mL) for 24 h. (F) Expression of miR-1233-3p in HUVECs treated PBS or growth factors (TGFβ, Ang-2, VEGF and FGF-2) for 24 h. Values are expressed as means± SEM and are expressed as fold change from PBS. * p < 0.05, compared with PBS.

Figure 2. Regulation of cell survival and migration by miR-1233-3p and Ang-1. (A,B) HUVECs transfected with control or miR-1233-3p mimics were maintained in complete medium, basic medium+FBS, or basic medium + Ang-1. (C,D) HUVECs transfected with control or miR-1233-3p inhibitors were maintained in complete medium, basic medium+ FBS, or basic medium + Ang-1. (E,F) Percentage wound healing and representative images of HUVECs transfected with control or miR-1233-3p mimics and treated with PBS or Ang-1 (300 ng/mL) for 8 h. (G) Percentage wound healing of HUVECs transfected with control or miR-1233-3p inhibitors and treated with PBS or Ang-1 (300 ng/mL) for 8 h. Values are means ± SEM. * p < 0.05, compared to PBS alone. # p < 0.05, compared to basal medium. ϕ p < 0.05, compared to cells transfected with control mimic or control inhibitor. Scale bar in panel F = 100 µm.

Figure 3. Effects of miR-1233-3p on EC differentiation and proliferation by miR-1233-3p. (A) The average tube lengths of capillary-like tube formation in HUVECs transfected with the control and miR-1233-3p mimic in the presence of PBS or Ang-1. Values are means ± SEM. (B) Representative images of capillary-like tube formation in cells transfected with control mimic in the presence of PBS (i) or Ang-1 (ii). Also shown are HUVECs transfected with the miR-1233-3p mimic in the presence of PBS (iii) or Ang-1 (iv). (C,D) The average and total tube lengths of cells transfected with the control and miR-1233-3p inhibitors in the presence of FBS or Ang-1. (E,F) BrdU incorporation in HUVECs transfected with the control and miR-1233-3p mimics or inhibitors in the presence of PBS. Values are means ± SEM. * p < 0.05, compared to PBS alone. ϕ p < 0.05, compared to cells transfected with control mimic or control inhibitor. Scale bar in panel B = 200 µm.

Figure 4. Identification of PDRG1 as miR-1233-3p target. (A) Venn diagram displaying in silico predicted targets of miR1233-3p as computed by DIANA, TargetScan, and miRDB algorithms. (B) PDRG1 mRNA expression in HUVECs transfected with miR-1233-3p mimic or inhibitor and their respective controls (denoted by dotted line). Values are expressed as fold change from control mimic and inhibitor. (C,D) Representative immunoblots of PDRG1 and β-ACTIN proteins and quantification of PDRG1 protein levels in HUVECs transfected with control or miR-1233-3p mimics. Values are means± SEM and are expressed as fold change from control mimic. (E,F) Representative immunoblots of PDRG1 and β-ACTIN proteins and quantification of PDRG1 protein levels in HUVECs transfected with control or miR-1233-3p inhibitors. Values are means± SEM and are expressed as fold change from control inhibitor. * p < 0.05, compared to control mimic or inhibitor.

Figure 5. PDRG1 is a direct target of miR-1233-3p. (A) A schematic representation of the sequence alignment of miR-1233-3p and the predicted binding sites according to the DIANA algorithm on the 3′ UTR of PDRG1 mRNA. The numbers between the brackets indicate the nucleotide number of 3’ UTR of PDRG1. (B–D) Biotinylated miR-1233-3p pull-down assays. (B,C) Illustrate the levels of miR-1233-3p and U6 RNA after the transfection of 50 nM biotinylated control or miR mimics in HUVECs. Values are expressed as a fold change from the control mimic. (D) Shows mRNA levels of PDRG1, HOXB3, and ZFP91 detected with qPCR in the pull-down materials isolated from HUVECs transfected with the biotinylated control or miR-1233 mimics. Values are expressed as a fold change from the control mimic. (E) The levels of PDRG1, HOXB3, and ZFP91 mRNA in the total input mRNAs measured by qPCR. Enrichment was calculated as follows: X = miR pull-down/control pull-down. Y = miR input/control input. Fold binding = X/Y. Values are expressed as means ± SEM. * p < 0.05, compared to the biotinylated control mimic.

Figure 6. Ang-1 regulates PDRG1 expression. (A,B) PDRG1 mRNA levels in HUVECs treated with PBS or Ang-1 (300 ng/mL) for 2, 4, 6, 10, 12, 24, 48, and 72 h. (C) Representative immunoblots of PDRG1 protein in HUVECs exposed to PBS or Ang-1 for 3, 6, 12, and 24 h. (D) Mean values of PDRG1 protein levels in HUVECs exposed to PBS or Ang-1 for 24 h. (E,F) Representative immunoblots of PDRG1 and β-ACTIN proteins and mean values of PDRG1 protein levels in HUVECs exposed for 24 h to PBS, Ang-2 (300 ng/mL), FGF-2 (10 ng/mL), or VEGF (40 ng/mL). Values are expressed as means± SEM and are expressed as a fold change from PBS. * p < 0.05, compared to PBS.