Chemerin-Induced Down-Regulation of Placenta-Derived Exosomal miR-140-3p and miR-574-3p Promotes Umbilical Vein Endothelial Cells Proliferation, Migration, and Tube Formation in Gestational Diabetes Mellitus

Figures

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  Figure 1. GDM differentially-expressed miRNAs. (A) Volcano plots show the fold change (log2) against the p value (−log10) of normal (n = 6) and GDM (n = 8) pregnant women. The significantly upregulated miRNAs (FC > 2) are depicted in red and the downregulated miRNAs (FC < 0.5) in blue. (B) Heatmap of the ten most changed miRNAs with medium and high abundance. (C) Scatter plot showing relative expression of the seven most changed miRNAs with medium and high abundance in placenta-derived exosomes from GDM (n = 30) versus those from normal (n = 30) pregnant women by qRT-PCR. ** p < 0.01, ns: no significance.

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  Figure 2. Effects of transfection of placenta-derived exosomes from normal and GDM women with miR-140-3p or miR-574-3p agomir or antagomir on the proliferation, migration, and tube formation of HUVECs in vitro. (A) Verification of placenta-derived exosomal miR-140-3p and miR-574-3p expression by qRT-PCR. EXOGDM transfected with miRNA agomir negative control (miRNA AG NC), miR-140-3p agomir (miR-140-3p AG), or miR-574-3p agomir (miR-574-3p AG). EXONor were transfected with miRNA antagomir negative control (miRNA Anta-AG NC), miR-140-3p antagomir (miR-140-3p Anta-AG), or miR-574-3p antagomir (miR-574-3p Anta-AG). (B) Immunofluorescence staining for placenta-derived exosomes marked with PKH26, (Scale bar: 20 µm). (C) The CCK-8 assay was applied to detect the proliferation of HUVECs after treatment with miRNA AG NC-EXOGDM, miR-140-3p AG-EXOGDM, miR-574-3p AG-EXOGDM, miRNA Anta-AG NC-EXONor, miR-140-3p Anta-AG-EXONor, or miR-574-3p Anta-AG-EXONor. (D) A transwell assay was applied to detect the migration of HUVECs after different treatments, (Scale bar: 20 µm). (E) A tube formation assay was applied to detect the tube formation of HUVECs after different treatments (n = 3, Scale bar: 100 µm). **…

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  Figure 3. Placenta-derived exosomal miR-140-3p and miR-574-3p expression was negatively regulated by chemerin. (A) Linear regression analysis of placenta-derived exosomal miR-140-3p expression and chemerin content in placenta tissues in pregnant women (n = 23). (B) Linear regression analysis of placenta-derived exosomal miR-574-3p expression and chemerin content in placenta tissues in pregnant women (n = 23). (C) ChemR23 protein levels in placenta tissues from control mice (Ctrl), chemerin-induced diabetic mice with injection of shRNA-negative control (CHM+sh-NC), and chemerin-induced diabetic mice with injection of chemR23 shRNA (CHM+sh-chemR23) detected by western blotting (n = 3). (D) Relative miR-140-3p and (E) miR-574-3p expression of placenta-derived exosomes from Control (EXOCtrl), CHM+sh-NC (EXOCHM+sh-NC) and CHM+sh-chemR23 (EXOCHM+sh-chemR23) mice measured with qRT-PCR (n = 3). ** p < 0.01.

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  Figure 4. Chemerin-induced placental inflammation-regulated trophoblast cell-derived exosomal miR-140-3p and miR-574-3p expression. (A) Immunofluorescence staining for F4/80 (macrophages) and chemerin of placenta tissue in normal and GDM pregnant women. (B) Content of IL-18 and IL-1β in placenta tissue of normal and GDM pregnant women was measured by ELISA (n = 3). (C) Content of IL-18 and IL-1β in placenta tissue of Ctrl, CHM+sh-NC, and CHM+sh-chemR23 mice was measured by ELISA (n = 3). (D) Protein levels of NOD-like receptor family pyrin domain containing 3 (NRLP3), apoptosis-associated speck-like protein containing CARD (Asc), pro caspase 3, cleaved caspase 3, pro caspase 9, cleaved caspase 9, pro caspase 1, cleaved caspase 1, pro-IL-1β, and pro-IL-18 in placental macrophages were measured by Western blotting. Protein levels of cleaved caspase 1, IL-1β, and IL-18 in the culture supernatants of placental macrophages were measured by Western blotting as well. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal control (n = 3). (E) Trophoblast cells were co-incubated with control (Ctrl), the supernatant from macrophage, macrophage+CHM+sh-NC, and…

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  Figure 5. miR-140-3p and miR-574-3p downregulate VEGF expression. (A) VEGF expression in the umbilical vein tissue of normal (n = 6) and GDM (n = 7) pregnant women was measured by Western blotting. (B) Linear regression analysis of miR-140-3p or miR-574-3p expression and VEGF in the umbilical vein tissue in pregnant women (n = 13). (C) VEGF expression of MUVECs after treatment with miRNA AG NC, miR-140-3p AG, miR-574-3p AG, miR-140-3p + miR-574-3p AG, miRNA Anta-AG NC, miR-140-3p Anta-AG, miR-574-3p Anta-AG, and miR-140-3p + miR-574-3p Anta-AG was measured by Western blotting (n = 3). (D) TargetScan and a protein–protein interaction (PPI) network were applied to identify the targets of miR-140-3p and their interaction. (E) Co-transfection of wild-type or mutant seed regions of VEGF 3′UTR were constructed with miRNA AG NC or miR-140-3p AG in MUVECs. A luciferase assay was applied to detect the luciferase activity (n = 3). (F) Protein levels of VEGF and hnRNP L after transfection with miRNA AG NC+vector and miR-574-3p AG+vector, miRNA AG NC+hnRNP L overexpression (OE), and miR-574-3p AG+hnRNPL OE were measured by Western blotting (n = 3). ** p < 0.01. ns: no significance.