miR-126a-3p induces proliferation, migration and invasion of trophoblast cells in pre-eclampsia-like rats by inhibiting A Disintegrin and Metalloprotease 9

Abstract

The present study aimed to investigate the underlying mechanism of miR-126a-3p in the proliferation, migration and invasion of trophoblast cells in pre-eclampsia-like rats by targeting A Disintegrin and Metalloprotease 9 (ADAM9). First, the interaction between miR-126a-3p and ADAM9 was confirmed via biochemical assays. Placental tissues and trophoblast cells were then obtained. RNA in situ hybridization was performed in order to detect miR-126a-3p expression in the placenta. Subsequently, a series of biological assays, including reverse transcription-quantitative PCR (RT-qPCR), Western blotting, MTT assay, apoptosis assay, cell cycle assay, wound healing assay and transwell assay were adopted in order to determine the cell proliferation, cell cycle distribution, apoptotic rate, and migration and invasion of trophoblast cells in each group. The results revealed that miR-126a-3p was down-regulated in the placenta of pre-eclampsia-like rats. In vivo experiments' results indicated that miR-126a-3p could inhibit ADAM9 expression, and induce cyclin D1, Matrix metalloproteinase (MMP) 2 (MMP-2), MMP-9 expression. MTT, apoptosis and cell cycle assay results revealed that trophoblast cells transfected with miR-126a-3p mimic or si-ADAM9 exhibited higher proliferative activity and a lower apoptotic rate compared with the blank group (all P<0.05). The wound healing assay and transwell assay results confirmed that, compared with the blank group, the migration and invasion ability of trophoblast cells in the miR-126a-3p mimic group and small interfering RNA (siRNA)-ADAM9 group were significantly increased (all P<0.05). Conversely, miR-126a-3p inhibitor treatment revealed the opposite effect (all P<0.05). In conclusion, the present study demonstrated that miR-126a-3p could enhance proliferation, migration and invasion, but decrease the apoptosis rate of trophoblast cells in pre-eclampsia-like rats through targeting ADAM9.

Keywords: ADAM9; biological characteristics; miR-126a-3p; preeclampsia; trophoblast cells.

Figure 1. Screening of key pre-eclampsia-associated miRNAs and genes via bioinformatics analysis

(A) Venn diagrams presenting the top ten differentially expressed miRNAs. The x-axis indicates the sample number, the y-axis presents the differentially expressed miRNAs. The key for the color scheme is present at the right panel with each rectangle corresponding to an individual sample. The red and blue colors indicate relatively high and low fold-change of expression, respectively. (B) miRWalk, miRSearch and TargetScan were used to identify pre-eclampsia-associated genes targeted by miR-126a-3p; PLXNB2, ADAM9, KANK2, PLK2, CRK, CAMSAP1 and PTPN9 were selected. (C) TargetScan predicts ADAM9 as an miR-126a-3p target gene.

Figure 2. Verification of miR-126a-3p target site on ADAM9

(A) The binding sites of miR-126a-3p and ADAM9 were verified using a luciferase reporter gene assay. HEK-293T cells were cultured in 96-well plates and then co-transfected with luciferase reporter WT or MUT plasmid, as well as NC or miR-126a-3p mimic sequence when cell confluence reached 80–90%. The luciferase activity was detected 48 h after transfection. (B) Verification of ADAM9 binding to Ago2 by RNA pull-down. Total protein was extracted from HEK-293T after cell lysis, followed by an RNA pull-down test according to the protocol of the kit. (C) Relative expression of proteins for RNA pull-down. (D) Total RNA was extracted from trophoblast cells 48 h after transfection, and the relative mRNA expression of miR-126a-3p and ADAM9 were determined via RT-qPCR analysis. (E) Bands of ADAM9 protein. (F) The total protein level of trophoblast cells in each group was extracted after 48 h transfection, and the relative protein expression of ADAM9 was determined via Western blotting. *P<0.05 vs. NC and ADAM9-3′UTR group; ^#P<0.05 vs. negative control group. The experiments were repeated in triplicate.

Figure 3. RNA in situ hybridization of placental tissue

Sample numbers of rats: pre-eclampsia, n=30; normal, n=10. The experiment was repeated three times.

Figure 4. Immunofluorescence staining of rat placental trophoblast cells

(A) Immunofluorescence profile of Alexa Fluor® 488-labeled cytokeratin 7 and vimentin expression in trophoblast cells. (B) Percentage of cytokeratin 7 and vimentin-positive cells.

Figure 5. Analysis reveals that stimulation of miR-126a-3p decreases ADAM9 but increases cyclin D1, MMP-2 and MMP-9 content in trophoblast cells with pre-eclampsia

(A,D) Relative mRNA expression of miR-126a-3p, ADAM9, cyclin D1, MMP-2 and MMP-9 were determined by RT-qPCR. (B) Protein bands of ADAM9, cyclin D1, MMP-2 and MMP-9 in the trophoblast cells of each group following transfection. (C,E) The relative protein expression of ADAM9, cyclin D1, MMP-2 and MMP-9 was determined via Western blotting. ^∧P<0.05 vs. Normal group; ^#P<0.05 vs. negative control group; ^&P<0.05 vs. miR-126a-3p mimic+siRNA-ADAM9 group. Sample numbers: Normal group, n=10; blank group, n=5; negative control, n=5; miR-126a-3p mimic group, n=5; miR-126a-3p inhibitor group, n=5; siRNA-ADAM9 group, n=5; and miR-126a-3p mimic+siRNA-ADAM9 group, n=5. The experiment was repeated three times.

Figure 6. Results of MTT assay indicate that the up-regulation of miR-126a-3p and ADAM9 silencing induces viability of trophoblast cells

After transfection for 48 h, the trophoblast cells were seeded into 96-well plates and MTT assay was applied in order to detect the cell viability in each group. ^∧P<0.05 vs. Normal group; ^#P<0.05 vs. negative control group; ^&P<0.05 vs. miR-126a-3p mimic+siRNA-ADAM9 group. Sample numbers: Normal group, n=10; blank group, n=5; negative control, n=5; miR-126a-3p mimic group, n=5; miR-126a-3p inhibitor group, n=5; siRNA-ADAM9 group, n=5; and miR-126a-3p mimic+siRNA-ADAM9 group, n=5. The experiment was repeated three times.

Figure 7. Up-regulation of miR-126a-3p and ADAM9 silencing relieves G₀/G₁ cell cycle arrest

(A) Distribution of cell cycle in each group after transfection. (B) Histogram describing the cell cycle in each group. ^∧P<0.05 vs. Normal group; ^#P<0.05 vs. negative control group; ^&P<0.05 vs. miR-126a-3p mimic+siRNA-ADAM9 group. Sample numbers: Normal group, n=10; blank group, n=5; negative control, n=5; miR-126a-3p mimic group, n=5; miR-126a-3p inhibitor group, n=5; siRNA-ADAM9 group, n=5; and miR-126a-3p mimic+siRNA-ADAM9 group, n=5. The experiment was repeated three times.

Figure 8. Up-regulation of miR-126a-3p and ADAM9 silencing inhibits the apoptosis of placental trophoblast cells

(A) Scatter diagram showing the cell apoptosis in each group. (B) Percentage of apoptotic cells in each group. ^∧P<0.05 vs. Normal group; ^#P<0.05 vs. negative control group ^&P<0.05 vs. miR-126a-3p mimic+siRNA-ADAM9 group. Sample numbers: Normal group, n=10; blank group, n=5; negative control, n=5; miR-126a-3p mimic group, n=5; miR-126a-3p inhibitor group, n=5; siRNA-ADAM9 group, n=5; and miR-126a-3p mimic+siRNA-ADAM9 group, n=5. The experiment was repeated three times.

Figure 9. Wound-healing assay results revealed that stimulation of miR-126a-3p induces migration of trophoblast cells in rats with pre-eclampsia

(A) Migration of trophoblast cells in each group after transfection for 48 h. (B) Percentage of migrated cells in each group. ^∧P<0.05 vs. Normal group; ^#P<0.05 vs. negative control group; ^&P<0.05 vs. miR-126a-3p mimic+siRNA-ADAM9 group. Sample numbers: Normal group, n=10; blank group, n=5; negative control, n=5; miR-126a-3p mimic group, n=5; miR-126a-3p inhibitor group, n=5; siRNA-ADAM9 group, n=5; and miR-126a-3p mimic+siRNA-ADAM9 group, n=5. The experiment was repeated three times.

Figure 10. Transwell assay results suggest that stimulation of miR-126a-3p induces invasion of trophoblast cells in rats with pre-eclampsia

(A) Migration of trophoblast cells in each group after transfection for 48 h. (B) Percentage of invasive cells in each group. ^∧P<0.05 vs. Normal group; ^#P<0.05 vs. negative control group; ^&P<0.05 vs. miR-126a-3p mimic+siRNA-ADAM9 group. Sample numbers: Normal group, n=10; blank group, n=5; negative control, n=5; miR-126a-3p mimic group, n=5; miR-126a-3p inhibitor group, n=5; siRNA-ADAM9 group, n=5; and miR-126a-3p mimic+siRNA-ADAM9 group, n=5. The experiment was repeated three times.

Figure 11. Molecular mechanism of miR-126-3p promotes the proliferation, migration and invasion of placental trophoblastic cells in pre-eclampsia rats through inhibiting ADAM9