Deficiency of DICER reduces the invasion ability of trophoblasts and impairs the pro‐angiogenic effect of trophoblast‐derived microvesicles

Figures

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  Figure 1. DICER knockdown impairs the invasion ability of both primary EVT and HTR8 cell line. A, Double staining of CK7 and HLA‐G by immunofluorescence to identify isolated human primary first‐trimester trophoblast (upper, 20×; lower, 40×). Scale bar = 40 µm. B, DICER mRNA levels in primary EVT relative to GAPDH determined by qPCR following transfection of control siRNA (si‐ctl) or DICER siRNA (si‐Dicer). The mean ± SEM is shown. **P < .01 (n = 3). C, Representative immunoblot of DICER in primary EVT following transfection of si‐ctl or si‐Dicer RNA. D, Invasion assay of HTR8 cells transected with si‐ctl or si‐Dicer RNA (top). Scale bar = 100 µm. Quantification of cell number at the outside membrane of transwell insert (bottom). The mean ± SEM is shown. **P < .01 (n = 3). E, Invasion assay of HTR8 cells transected with sh‐scr or sh‐Dicer LVs (top). Scale bar = 200 µm. Quantification of cell number at the outside membrane of transwell insert (bottom). The mean ± SEM is shown. ***P < .001 (n = 3). F, Invasion assay of HTR8 cells with the treatment of 10 mmol/L enoxacin. Scale bar = 200 µm. Quantification of cell number at the outside membrane of transwell insert (bottom). The mean ± SEM is…

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  Figure 2. Increased expression of miR‐16‐2‐3p is responsible for the reduced invasion ability of sh‐Dicer HTR8 cells. A, Heat map of miRNAs by miRNA sequence in sh‐scr and sh‐Dicer HTR8 cells. miRNAs of high abundance and significant change are underlined. High relative expression in light red and low relative expression in dark red. B, miRNA levels (including let‐7a, let‐b, let‐7d, let‐7e and miR‐16‐2) relative to U6 determined by qPCR in sh‐scr and sh‐Dicer HTR8 cells. The mean ± SEM is shown. *P < .05, **P < .01, ns, non‐significant (n = 3). C, invasion assay of sh‐scr HTR8 cells transfected with miRNA inhibitor control (inhibitor ctl) and sh‐Dicer HTR8 cells transfected with miRNA inhibitor ctl or miR‐16‐2‐3p inhibitor (left). Scale bar = 200 µm. Quantification of cell number at the outside membrane of transwell insert (right). The mean ± SEM is shown. *P < .05, **P < .01 (n = 3). D, Invasion assay of HTR8 cells transfected with miRNA mimic control (mimic ctl) or miR‐16‐2‐3p mimic (left). Scale bar = 200 µm. Quantification of cell number at the outside membrane of transwell insert (right). The mean ± SEM is shown. *P < .05, ***P < .001 (n = 3)

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  Figure 3. COL1A2 is a target of miR‐16‐2‐3p and mediates the impaired invasion of sh‐Dicer HTR8 cells. A, Representative immunoblot of COL1A2 in sh‐scr and sh‐Dicer HTR8 cells. TUBULIN was blotted as loading control. B, Invasion assay of HTR8 cells transfected with si‐ctl or si‐Col1a2 RNA (left). Scale bar = 200 µm. Quantification of cell number at the outside membrane of transwell insert (right). The mean ± SEM is shown. ***P < .001 (n = 3). C, Representative immunoblot of COL1A2 in sh‐scr HTR8 cells transfected with miRNA inhibitor ctl and sh‐Dicer HTR8 cells transfected with inhibitor ctl or miR‐16‐2‐3p inhibitor. TUBULIN was blotted as loading control. D, Representative immunoblot of COL1A2 in HTR8 cells transfected with miRNA mimic ctl or miR‐16‐2‐3p mimic. TUBULIN was blotted as loading control. E, Schematic diagram shows one predicted binding site of miR‐16‐2‐3p on the wild‐type 3′‐UTR of COL1A2 (WT) where red bases in miR‐16‐2‐3p represent its seed region. Five bases underlined by green line in WT are changed into five other bases shown in green colour and referred as to MUT. F, Relative luciferase activity (firefly luminescence/Renilla luminescence) of HTR8 cells 48 h after…

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  Figure 4. DICER knockdown in HTR8 cells impairs the pro‐angiogenetic effect of microvesicles on HUVECs. A, Tube formation assay of HUVECs treated with HTR8 CM or DMEM. Scale bar = 200 µm. Quantification of tube length formed by HUVECs (right). The mean ± SEM is shown. **P < .01, ns, non‐significant (n = 3). B, Tube formation assay of HUVECs treated with different concentration of HTR8 MVs. Scale bar = 200 µm. Quantification of tube length formed by HUVECs (right). The mean ± SEM is shown. **P < .01, ns, non‐significant (n = 3). C, Tube formation assay of red cell tracker stained HUVECs cocultured with green cell tracker stained sh‐scr or sh‐Dicer HTR8 cells at the ratio of 2:1 (left). Scale bar = 130 µm. Quantification of tube length formed by HUVECs (right). The mean ± SEM is shown. **P < .01, (n = 3). D, Tube formation assay of HUVECs treated with sh‐scr or sh‐Dicer CM supplemented with 2% FBS (left). Scale bar = 200 µm. Quantification of tube length formed by HUVECs (right). The mean ± SEM is shown. **P < .01, (n = 3). E, Tube formation assay of HUVECs treated with purified sh‐scr or sh‐Dicer MVs resuspended in DMEM supplemented with 2% FBS (left). Scale bar = 200 µm. Quantification of…

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  Figure 5. Reduced VEGFA, DICER protein and increased miR‐16‐2‐3P and let‐7d‐5p were found in the sh‐Dicer CM and sh‐Dicer MVs, respectively. A, Representative immunoblotting of DICER in sh‐scr and sh‐Dicer MVs. TUBULIN is used as loading control. B, miRNAs levels (let‐7d‐5p, miR‐16‐2‐3p and miR‐15b) relative to U6 determined by qPCR in sh‐scr and sh‐Dicer HTR8 MVs). The mean ± SEM is shown. *P < .05, **P < .01, (n = 3). C, Tube formation assay of HUVECs treated with HTR8 MVs suspended in DMEM with different treatment (A: HTR8 MVs only; B: HTR8 MVs supplemented by DICER Ab; C: HTR8 MVs electroporated with DICER Ab; and D: HTR8 MVs electroporated with IgG). Scale bar = 300 µm. Quantification of tube length formed by HUVECs (right). The mean ± SEM is shown. **P < .01, (n = 3). D, Human angiogenesis array of sh‐scr and sh‐Dicer CM. Some angiogenesis‐related factors with significant change of expression are highlighted (right). Normalized relative intensity of black dot of angiogenic factors (left). The mean ± SEM is shown. **P < .01, (n = 2). E, Quantification of VEGFA protein concentration in sh‐scr and sh‐Dicer CM by ELISA. The mean ± SEM is shown. ***P < .001, (n = 3). F, VEGFA protein…

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  Figure 6. Decreased expression of DICER protein in PE placenta. A, Representative immunoblot of DICER in placenta from PE, sPE patients and control normotensive pregnant women (CTL) at delivery. TUBULIN was blotted as loading control (top). Intensity ratio of DICER to TUBULIN (lower left) in CTL group, sPE group and PE group. The mean ± SEM is shown. **P < .01 and ***P < .001 (n = 10 for CTL, n = 6 for PE and n = 6 for sPE). B, DICER mRNA levels relative to GAPDH in placenta from PE, sPE patients and normotensive pregnant women at delivery determined by qPCR. The mean ± SEM is shown. ns, non‐significant, (n = 10 for CTL, n = 6 for PE and n = 6 for sPE). C, DICER immunohistochemistry‐stained placental sections from sPE patients and normotensive pregnant women where red star labels CTB, yellow asterisk labels STB and green arrow labels endothelial cells. Scale bar = 150 µm

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  Figure 7. Schematic diagram shows the proposed mechanism of DICER mediating the invasion ability of HTR8 cells and the crosstalk between HTR8 cells and HUVECs during spiral artery remodelling in an in vitro model. DICER plays an important role in balancing the expression of miR‐16‐2 and let‐7 family, and there seems to be a positive feedback between DICER and let‐7 family where DICER inhibits the transcription of MIRLET7A1 gene and DICER is a target of let‐7a (49). Increased level of miR‐16‐2‐3p leads to decreased invasion ability and VEGFA secretion by targeting COL1A2 and VEGFA, respectively, in HTR8 cells following DICER knockdown. Silencing of DICER also creates an anti‐angiogenic bias in the extracellular microenvironment of HTR8 cells with dysregulated angiogenic factors (up‐regulated IGFBP3, down‐regulated VEGFA, HB‐EGF, MMP9 and ANGIOGENITIN), and increased miRNAs (miR‐16‐2, miR‐15b and let‐7d) and decreased DICER protein in HTR8 cell‐derived MVs, leading to impaired angiogenesis of HUVECs