miR-381-3p contribution in mouse spontaneous abortion via targeting VEGFA

Animal model and miRNA-seq

Eight-week-old female and male C57BL/6 mice were obtained from Shanghai Lingchang Biotechnology Co., Ltd. (Shanghai, China). The mice were kept in the Laboratory Animal Center of Nanjing Ramda Pharmaceutical Co., Ltd. (Nanjing, China) and lived in CP-4 cages (L × W × H: 290 × 178 × 160 mm). The mice density of each cage did not exceed six. The mice were allowed to eat and drink freely at a temperature of 24 ± 2 °C, relative humidity of 40–60%, and a light-dark cycle for 12 h. After acclimatization for 1 week, ten female mice in proestrus were randomly divided into two groups: Control and abortion. As previously reported (Ding & Lu, 2014), after carbon dioxide (CO₂) anesthesia, each animal in the abortion groups was intraperitoneally injected with 10 µg beta2-glycoprotein I (β2-GPI) (Abcam) on the 1^st day and immunized once on the 8^th day. The female mice in the control group were intraperitoneally injected with normal saline. On the 18^th day, the female mice were mated with male mice at a ratio of 1:2. The presence of pessaries was considered to be on the 0.5^th day of pregnancy. On the 15^th day of pregnancy, the animals were euthanized using CO₂, and the placenta tissues of the two groups were frozen in liquid nitrogen. As previously described (Li et al., 2024), miRNA-seq was used to detect miRNA expression profiles in the placentas of the two groups (n = 3). miRNA-seq and analysis of differentially expressed miRNAs were conducted by Beijing Boao Crystal Code Biotechnology Co., Ltd. (Beijing, China).

The animal experiment was approved by the Institutional Animal Care and Use Committee of Nanjing Ramda Pharmaceutical Co., Ltd. (No. IACUC-20230208) and performed following the Animal Care Committee.

Cell culture

C166 cells (a mouse vascular endothelial cell line) were acquired from Beijing Baiou Bowei Biotechnology Co., Ltd. (Beijing, China). The cells were cultured in Dulbecco’s Modified Eagle Medium with 10% fetal bovine serum (Life Technologies, Carlsbad, CA, USA) and 1% penicillin/streptomycin in an atmosphere of 5% CO₂ and 95% air at 37 °C.

Cell transfection

miR-381-3p mimics, miR-381-3p inhibitor, VEGFA siRNA, and negative control (NC) were synthesized by Jiangsu KeyGEN Biotechnology Co., Ltd. (KeyGEN BioTECH, Nanjing, China). They were respectively transfected into C166 cells for 24 h using Lipofectamine™ 3000 (Invitrogen, Waltham, MA, US) following the manufacturer’s instructions. All experiments were repeated thrice. The sequences used are listed in Table 1.

Wound healing assay

A straight line was drawn in the 6-well plate using a 200 μL yellow sterile pipette tip when the cell fusion degree had reached 80–90%. The cell migration was photographed through an inverted microscope (Olympus, Shinjuku City, Tokyo, Japan) at 0 and 24 h following transfection.

Transwell assay

Transwell assay was used to measure C166 cell migration. The transfected cells were added to the upper chamber of the Transwell, and a complete medium was added to the 24-well plate. After 24 h of culture, the medium in each well was removed. The cells in the Transwell chambers were fixed with methanol for 30 min. The upper cells of the microporous membrane in the Transwell chamber were removed using a cotton swab, and the lower cells were stained with 0.1% crystal violet (Sigma, Kanagawa, Japan) for 20 min. The migratory cells were photographed and counted using an inverted microscope (Olympus, Shinjuku City, Tokyo, Japan).

Tube formation assay

The angiopoiesis of C166 cells was examined using a tube formation assay. The cells were digested with 0.25% trypsin-EDTA (KeyGEN BioTECH, San Francisco, California, US) 24 h after transfection. Briefly, 200 µL of cell suspension (4 × 10⁵ cells/mL) was added to per well in a 96-well plate coated with Matrigel (BD Biosciences, San Jose, CA, USA). After incubation at 37 °C for 6 h, angiogenesis was observed using an IX51 microscope (Olympus, Shinjuku City, Tokyo, Japan). The total branch points and capillary length were counted in three random fields of view using Gel-Pro32 software.

Dual-luciferase reporter assay

The wild-type (WT) and mutant-type (MUT) VEGFA sequences were designed following the binding sites of miR-381-3p and VEGFA 3′ UTR predicted by the StarBase database (https://rnasysu.com/encori/). VEGFA WT and MUT sequences (the 200 bp upstream and downstream of chr17:46017343-46017348) were inserted into pmirGLO vectors to construct pmirGLO-VEGFA-WT and pmirGLO-VEGFA-MUT recombinant vectors. The Dual-Glo® Luciferase Assay System (Promega, Madison, WI, USA) was used to determine the reporter activities. In a 96-well black plate, pmirGLO-VEGFA-WT or pmirGLO-VEGFA-MUT was co-transfected with the miR-381-3p mimic or NC into 293T cells using Lipofectamine™ 3000 (Invitrogen, Waltham, MA, USA). After transfection for 48 h, 50 µL of lysed cells were added to each well. Afterward, the Dual-Glo® Luciferase Reagent and Dual-Glo® Stop & Glo® Reagent were added to each well in turn. The fluorescence densities were detected by a Tecan Spark microplate reader (Tecan, Männedorf, Switzerland). The luciferase activities were calculated using the ratio of firefly/renilla activities.

miR-381-3p knockdown in vivo

As described in the animal model construction above, 15 eight-week-old female and male C57BL/6 mice in proestrus were randomly divided into three groups: Control, abortion, and abortion+shmiR-381-3p. On the 9^th day of β2-GPI treatment, each animal in the abortion and abortion+shmiR-381-3p groups was intraperitoneally injected with 2 × 10¹¹ vg of adeno-associated virus (AAV)-shNC or AAV-shmiR-381-3p synthesized from Guangzhou PackGene Biotechnology Co., Ltd. The female mice in the control group were intraperitoneally injected with normal saline. On the 18^th day, female mice were mated with male mice. On the 15^th day of pregnancy, the mice were euthanized by CO₂, and the weight of the fetus and placenta was counted and photographed. The placenta was carefully divided into two parts: One was fixed with 4% paraformaldehyde, and the other was frozen with liquid nitrogen and then stored at –80 °C.

Total RNA extraction and real-time quantitative polymerase chain reaction (RT-qPCR)

The total RNA of the cells and placentas was extracted with TRIzol Reagent (Invitrogen, Waltham, MA, USA). The purity of the total RNA was determined using an ultraviolet-visible spectrophotometer (UV-2450; SHIMADZU, Kyoto, Japan). PrimeScript^TM RT master mix (Takara, Shiga, Japan) was added for reverse transcription of total RNA. For miRNA analysis, the first strand of miRNA cDNA was obtained using the stem-loop method. SYBR Green qPCR Mix (Takara, Shiga, Japan) was used to examine the gene expression. The PCR reaction procedure, including holding stage (95 °C for 5 min), 40 cycles (95 °C for 20 s, 60 °C for 20 s, and 72 °C for 20 s), and melt curve stage (95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s), was performed using the LightCycler480 II real-time fluorescence quantitative PCR instrument (Roche, Basel, Switzerland). GAPDH or U6 was used as an internal reference. The relative expression levels of miR-381-3p and Vegfa were calculated using the 2^−ΔΔCt method. Three biologically independent assays were performed. The primer sequences of miR-381-3p, Vegfa, Gapdh, and U6 were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China), as listed in Table 2.

Western blotting

The cell and placenta samples were treated with EBC buffer (Roche Diagnostics, Basel, Switzerland, USA) and 1 mM phosphatase inhibitor mixture II (Sigma-Aldrich, St. Louis, MO, USA). SDS-PAGE was performed by transferring proteins onto nitrocellulose membranes. Subsequently, 5% non-fat milk was used to block the membranes. The membranes were then incubated overnight at 4 °C with primary antibodies, including VEGFA (19003-1-AP; Proteintech, Rosemont, IL, USA, 1:500), p65 (ab16502; Abcam, Cambridge, USA, 1:1,000), p-p65 (ab76302; Abcam, Cambridge, USA, 1:1,000), and GAPDH (ab9485; Abcam, Cambridge, USA, 1:2,000). After rinsing with 1× TBST; the membranes were incubated with the respective secondary antibodies conjugated with horseradish peroxidase for 1 h at room temperature. The protein bands were visualized using Immobilon™ Western Chemiluminescent HRP Substrate (Cat. No.: WBKLS0500; Millipore Corporation, Burlington, MA, USA), and the images were captured on the visualization instrument Tanon-5200 (Tanon, Xinjiang, China).

Hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC) assay

On the 15^th day of pregnancy, the placenta samples were fixed in a 4% paraformaldehyde solution and embedded in paraffin. The paraffin-embedded placental tissues were sectioned to a thickness of 4 μm, deparaffinized with xylene, and rehydrated in a graded series of ethanol. H&E staining was performed, and antigen retrieval was conducted by microwaving in the citric acid buffer. The sections were incubated with antibodies against CD31 (ab182981; Abcam, Cambridge, USA, 1:500), rinsed, and incubated with a secondary antibody for 60 min at room temperature. Three randomly selected visual fields were analyzed using Image-Pro Plus software (version 6.0).

Enzyme-linked immunosorbent assay (ELISA)

The blood of each mouse was obtained from the eye socket and centrifuged at 3,000×g, 4 °C for 20 min. The concentrations of serum total proteins were detected by a BCA assay kit (KeyGEN BioTECH, Nanjing, China). The levels of interleukin (IL)-10, tumor necrosis factor α (TNF-α), homocysteine (HCY), annexin V (ANX-V), and methylene tetrahydrofolate reductase (MTHFR) were measured using an ELISA Kit (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China) under a Tecan Spark microplate reader (Tecan, Männedorf, Switzerland).

Statistical analyses

All data are presented as mean ± standard deviation (SD) of at least three biological replicates per experiment. The data were statistically analyzed using the Statistical Package for the Social Sciences (SPSS) software (version 22.0). A one-way analysis of variance was used to analyze differences among groups using the LSD method. A P < 0.05 indicated statistical significance.

miR-381-3p inhibited the migration and angiogenesis in C166 cells

To explore potential miRNAs that regulate angiogenesis in the placental tissue of RSA, miRNA-seq was used to identify differentially expressed miRNA profiles in normal and abortion-pregnant mice. The results demonstrated that 133 miRNAs were upregulated, and 24 miRNAs were downregulated in the placental tissue of the abortion group than the normal group (Figs. 1A, 1B and Table S1). Among the upregulated miRNAs, miR-381-3p bound to VEGFA with a higher binding score (Fig. 1C), according to multiple miRNA databases (StarBase, miRwalk, TargetScan, and miRTarBase) (Jiang et al., 2024; Zhao et al., 2025). This suggests that miR-381-3p may affect placental angiogenesis by regulating VEGFA. The miRNA-seq raw data were uploaded to the Gene Expression Omnibus Datasets (Accession: GSE286463).

Subsequently, we examined whether miR-381-3p regulated angiogenesis in vitro. As presented in Fig. 2, miR-381-3p mimics significantly inhibited the migration of C166 cells (P < 0.001) (Figs. 2A–2C) and reduced branch points and capillary length during angiogenesis (P < 0.01) (Figs. 2A, 2D and 2E), compared with the NC group. However, miR-381-3p inhibitor revealed the opposite effect (Figs. 2C–2G). These findings demonstrated that miR-381-3p suppressed the migration and angiogenesis in C166 cells.

miR-381-3p suppressed the activation of the VEGFA/NF- $\text{κ}$B pathway

Activation of the VEGFA/NF-κB pathway is essential for angiogenesis (Lin et al., 2023; Mantsounga et al., 2022). In this study, miR-381-3p mimics significantly reduced Vegfa mRNA expression in C166 cells (P < 0.01), whereas miR-381-3p knockdown increased Vegfa mRNA expression (P < 0.001) (Figs. 3A and 3B). Notably, miR-381-3p mimics inhibited the expression levels of the VEGFA and p-p65 proteins (Fig. 3C). These findings indicated that miR-381-3p may inhibit angiogenesis in C166 cells by regulating the activation of the VEGFA/NF-κB pathway.

miR-381-3p suppressed the migration and angiogenesis of C166 cells through targeting VEGFA

As presented in Fig. 4A, a dual-luciferase reporter system was used to identify the binding of miR-381-3p and VEGFA 3′ UTR. The results demonstrated that miR-381-3p mimics significantly decreased the relative fluorescence activity in the VEGFA-WT group but had no effect on the fluorescence activity in the VEGFA-MUT group (Fig. 4A). Subsequently, three siRNA sequences of VEGFA were constructed. RT-qPCR verification revealed that VEGFA siRNA#2 most significantly decreased Vegfa mRNA expression (Fig. 4B). As a result, VEGFA siRNA#2 was used to silence Vegfa mRNA expression. VEGFA silence reversed the effect of the miR-381-3p inhibitor on C166 cell migration and angiogenesis. These results revealed that miR-381-3p inhibited the migration and angiogenesis of C166 cells by targeting VEGFA.

miR-381-3p knockdown partially rescued abortion by regulating vascular remodeling, inflammation, and RSA-related factors

To explore the role of miR-381-3p in the placenta and fetus in the abortion mouse model, AAV-mediated shmiR-381-3p was constructed and injected intraperitoneally into pregnant mice to achieve miR-381-3p knockdown. In the control group, the placenta and fetus were intact; however, the abortion mice had fetal deformities and lower weights of the placenta and fetus (Fig. 5A). miR-381-3p knockdown improved the fetal status of the abortion model, including observable limbs and increased weight of placenta and fetus, compared with the abortion group (Fig. 5A). Moreover, CD31 positive expression in the abortion group was significantly lower than that in the control group (P < 0.01), while CD31 expression in the abortion+shmiR-381-3p group was significantly higher than that in the abortion group (P < 0.05) (Fig. 5B), suggesting that miR-381-3p knockdown may promote angiogenesis in the placenta to improve ischemic/anoxic state of the placenta in the abortion mice. HE staining results revealed that the decidual cells were abundant, with scattered inflammatory cells; the vascular structure was normal, and trophoblast cells were visible without vascular necrosis. In the abortion group, the decidual cells decreased, and the blood vessel wall thickened, accompanied by vascular necrosis. In the abortion+shmiR-381-3p group, the decidual cells were abundant, and vascular thickening or necrosis was not observed (Fig. 5C).

Compared with the control group, the expression level of Vegfa mRNA decreased significantly in the abortion group (P < 0.001), whereas miR-381-3p knockdown reversed this result (Fig. 6A). The expression levels of VEGFA and p-p65 proteins decreased significantly in the abortion group compared with the control group (P < 0.01), while miR-381-3p knockdown increased the expression levels of VEGFA and p-p65 proteins than the abortion group. The levels of IL-10, ANX-V, and MTHFR in the abortion group were significantly lower than those in the control group (P < 0.001), whereas miR-381-3p knockdown significantly increased the levels of the above three factors, compared with the abortion group (P < 0.01) (Figs. 6C–6E). TNF-α and HCY levels revealed opposite results (Figs. 6F and 6G). Elevated HCY level induces endothelial cell damage to increase uterine artery resistance in the non-pregnant state, which leads to an increased risk of RSA (Yang et al., 2023). ANX-V can maintain a normal pregnancy by preventing blood clotting and may play a beneficial role in RSA (Murad et al., 2023). Furthermore, the inactivation or absence of MTHFR leads to HCY accumulation, increasing the risk of RSA (Lei et al., 2024; Zhang, Fu & Wei, 2021). These findings suggest that miR-381-3p knockdown can rescue abortion by promoting angiogenesis, inhibiting inflammation, and regulating RSA-related factors.