Human placental tissue used in this research was collected from 38 patients that had provided written informed consent. The present study was approved by the Ethics Committee of Zhongnan Hospital of Wuhan University (Wuhan, China; permit no. 2016016).

Human placental tissues were collected from artificial labor and caesarean sections at the Renmin Hospital of Wuhan University (Wuhan, Hubei, China) between June 2016 and February 2017. All samples were frozen in liquid nitrogen for 5 min and stored at −80°C for subsequent analyses. Placental tissue was collected from pregnant women who met the following inclusion criteria: Monocyesis, normal pregnancy at the time of sample collection and no pre-existing clinical conditions, including diabetes, chronic nephritis, cardiovascular, hepatic or autoimmune disease. Samples were subsequently categorized into the PE or normal group according to the presence or absence of PE. Detailed clinical characteristics of the sample groups are presented in Table I.

Placental tissue from different treatment groups was sequentially fixed in 4% paraformaldehyde at room temperature for 12 h, dehydrated in 60, 70, 80, 90, 95% and absolute ethanol respectively for 30 min at room temperature, embedded in paraffin and cut into 5 µm sections. Following deparaffinization in xylene for 5 min twice and rehydration sequentially in absolute, 95, 90, 80, 70 and 60% ethanol respectively for 5 min at room temperature, placental tissue sections were stained with H&E. Images were captured under ×100 magnification by a Nikon Eclipse Ci microscope (Nikon Corporation, Tokyo, Japan).

Immunohistochemical detection of VEGFA was conducted on 5-µm-thick paraffin-embedded placental tissue sections using the avidin-streptavidin-biotin method. IHC operation kits was purchased from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). The standard IHC protocol was performed according to the manufacturer's instructions. Following blocking in immunnol staining blocking buffer (P0102; Beyotime Institute of Biotechnology) at room temperature for 1 h and incubation with a human VEGFA primary antibody (1:50; ab1316; Abcam, Shanghai, China) at 4°C overnight, sections were washed with PBS and subsequently incubated with the biotin-conjugated goat anti-mouse IgG (TA130008; 1:200; OriGene Technologies, Inc., Beijing, China) at room temperature for 45 min. Visualization was achieved with diaminobenzidine staining (OriGene Technologies, Inc., Beijing, China) for 5 min and counterstaining with hematoxylin solution for 2 min at room temperature. Images were captured with the Nikon Eclipse Ci microscope (Nikon Corporation, Tokyo, Japan) at ×100 magnification.

Each human placental tissue sample for RT-qPCR was obtained from the center between placental edge and umbilical cord. The size of the samples was ~1.5×1.5×1.5 cm³. Total RNA was isolated with TRIzol reagent (Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. Quantitative real-time PCR kits were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Details of the RNA extraction, cDNA conversion and RT-qPCR assays have been described previously (23). To detect the mRNA expression levels of miR-203 and VEGFA, RT-qPCR was performed in 96-well reaction plates with a total volume of 10 µl, containing the following: 1 µl cDNA template, 0.2 µl each primer, 3.6 µl diethyl pyrocarbonate-H₂O and 5 µl SYBR-Green dye using the ABI Step One Plus™ Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following primers were designed using Primer Premier version 6.0 (Premier Biosoft International, Palo Alto, CA, USA): miR-203 forward, 5′-GAATTCGGGGATCTGGGCGCAGGGGCC-3′ and reverse, 5′-CTCGAGCCGACCTGGAGCGCGGAGC-3′; VEGFA forward, 5′-ACCATGAACTTTCTGCTGTCTTGGGTGCAT-3′ and reverse, 5′-TCACCGCCTCGGCTTGTCACATCTGCAAGT-3′; GAPDH forward, 5′-TTGATGGCAACAATCTCCAC-3′ and reverse, 5′-CGTCCCGTAGACAAAATGGT-3′; and 6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. The PCR cycling parameters used were as follows: Pre-denaturation at 95°C for 30 sec; 40 cycles of denaturation at 95°C for 5 sec, annealing at 60°C for 10 sec and extension at 72°C for 30 sec. GAPDH and U6 mRNA expression levels were measured and applied as quantitative controls to normalize the relative expression of VEGFA and miR-203, respectively. The relative expression of each target amplicon was calculated using the 2^−ΔΔCq method relative to the endogenous control (24).

The HTR-8/SVneo cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). This cell line was established by the transfection of first trimester villous explants of human primary trophoblasts. Cells were cultured in RPMI 1640 (11875-093; Thermo Fisher Scientific, Inc.) medium supplemented with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) without antibiotics for 24 h. miR-203 mimic (Thermo Fisher Scientific, Inc.), miR-203 inhibitor (anti-miR-203; Thermo Fisher Scientific, Inc.) and a scramble control miRNA (Shanghai GenePharma Co., Ltd., Shanghai, China) were subsequently transfected into cells seeded on a 6-well plate at 10,000 cells/well, as described previously (21). Sequences were transfected at a final concentration of 10 nM using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc., Shanghai, China) according to the manufacturer's protocol. This concentration was determined based on dose-response experiments. Cells were further cultured for the following cell proliferation and transwell assays and subsequently harvested for RT-qPCR or western blot analysis 48 h after transfection. All experiments were conducted in triplicate.

For the cell proliferation assay, transfected HTR-8/SVneo cells were seeded into 96-well plates (5×10⁴ cells/well). Cells were cultivated in RPMI 1640 medium for 3 days. CCK-8 (10 µl; American Type Culture Collection; Manassas, VA, USA) was subsequently added to each well and HTR-8/SVneo cells were incubated for 2 h. Cell viability was measured by the absorbance at a wavelength of 450 nm using an ELISA reader (Tecan Group, Ltd., Mannedorf, Switzerland).

Cell migration and invasion was detected using Transwell permeable supports (Corning Incorporated, Corning, NY, USA). HTR-8/SVneo cells were transfected with the miR-203 mimic, anti-miR-203 or the negative control (miRNC) for 24 h and subsequently cultured in 200 µl of serum-free RPMI 1640 prior to transfer onto the upper chambers of 24-well plates (5×10⁵ cells/well) with or without a Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) coating. The lower chamber was supplemented with 800 µl of RPMI 1640 with 10% FBS. After 24 h without Matrigel or 48 h with Matrigel, cells in the lower chamber were fixed with absolute methanol for 20 min and stained with 0.1% crystal violet solution (Sigma Aldrich; Merck KgaA, Darmstadt, Germany) for 10 min at room temperature prior to cell counting under a microscope.

Western blot analysis was performed as described previously (21). Total protein was extracted using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) and Protease Inhibitor Cocktail (Sigma Aldrich; Merck KgaA). A BCA Assay Kit (Thermo Fisher Scientific, Inc.) was applied to detect the concentration of extracted protein. A total of 30 µg was loaded onto each lane, separated using 10% SDS-PAGE and blotted onto polyvinylidene difluoride membranes (Merck KgaA). The membrane was subsequently blocked in 5% non-fat milk for 1 h at room temperature and incubated overnight at 4°C with the primary antibodies VEGFA (1:1,000; ab1316, Abcam) and GAPDH (sc-47724; 1:3,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Following incubation with horseradish peroxidase-conjugated anti-rabbit (A0208; 1:5,000; Beyotime Institute of Biotechnology) and anti-mouse (A0216; 1:5,000; Beyotime Institute of Biotechnology) secondary antibodies at room temperature for 45 min, antibody binding signals were detected using the enhanced chemiluminescence luminol reagent (PerkinElmer Inc., Waltham, MA, USA) and a Chemi-doc image analyzer (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The protein bands were subsequently visualized with ImageJ software version 1.49 (National Institutes of Health, Bethesda, MD, USA). GAPDH was used to normalize the band intensities.

The luciferase reporter assay was conducted as described previously (21). The 3′-untranslated region (UTR) sequence of VEGFA was amplified and inserted into the luciferase reporter vector pGL3-enhancer (Promega Corporation, Madison, WI, USA). The primers for VEGFA 3′-UTR were 5′-CAGCTCGAGTGTGTGAGTGGTTGACCTTCCT-3′ (forward) and 5′-CCGAAGCTTTCAGGGAGAGAGAGATTGGAAA-3′ (reverse). HTR-8/SVneo cells were seeded into 24-well plates (5×10⁵ cells/well) and incubated for 24 h. Wild-type or mutant VEGFA 3′-UTR vectors were subsequently co-transfected with the miR-203 mimic into HTR-8/SVneo cells using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). After incubation for 48 h, cells were lysed and assayed for luciferase activity with the Luciferase Assay System (Promega Corporation) according to the manufacturer's instructions. Renilla-luciferase was used for normalization.

Data analysis was performed with the Stata 13.0 software (StataCorp LP, College Station, TX, USA), using the Student's t-test, one way analysis of variance and the Scheffe post hoc test with Spearman's correlation analysis. Results are presented as the mean ± standard error. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the role of miR-203 and VEGFA in the placenta development, miR-203 and VEGFA expression levels in all 38 placental tissue samples were measured using RT-qPCR. miR-203 expression levels were significantly increased in PE placentas compared with normal placentas (Fig. 1A; P<0.01), whereas VEGFA expression levels were significantly decreased (Fig. 1B; P<0.01). The correlation between miR-203 and VEGFA expression was further evaluated and a significant negative correlation (r=0.2217; P=0.0028; Fig. 1C) was identified. These results indicate that miR-203 upregulation may be associated with VEGFA downregulation.

To confirm morphological changes in the PE placenta, H&E staining was applied to the normal and PE placenta tissue samples. A disordered structure and a prominent decrease in chorionic villi was observed in the PE placenta in comparison with normal placenta tissue (Fig. 2A and B). IHC revealed abundant VEGF expression in villous trophoblasts and a lack of expression in the villous endothelium. VEGFA expression levels in the PE placentas was clearly downregulated in comparison with normal placenta samples (Fig. 2C and D). These results suggest that VEGFA downregulation in the placenta may be associated with PE occurrence.

The HTR-8/SVneo cell line is established by the transfection of human primary trophoblasts originating from first trimester villous explants, with a gene encoding the simian virus 40 large T antigen, resulting in immortalization (25). To determine the effect of miR-203 on cell proliferation in the human placenta, HTR-8/SVneo cells were transiently transfected with miR-203 mimics and anti-miR-203, and a CCK-8 assay was performed to test the cell proliferation rate. RT-qPCR was used to confirm the overexpression or inhibition of miR-203 expression in HTR-8/SVneo cells (Fig. 3A). Overexpression of miR-203 was demonstrated to reduce the proliferative capacity of HTR-8/SVneo cells, whereas the inhibition of miR-203 expression resulted in a significant increase in cell proliferation (Fig. 3B). To assess the effects of miR-203 on cell migration and invasion, Transwell migration and invasion assays were performed in the HTR-8/SVneo cells. Overexpression of miR-203 significantly suppressed the migration and invasion abilities of HTR-8/SVneo cells compared to the control cells (Fig. 4A and B). The migration and invasion capacity of HTR-8/SVneo cells in the anti-miR-203 group was also significantly enhanced.

To evaluate the possible effect of miR-203 on VEGFA expression, VEGFA mRNA and protein expression levels in HTR-8/SVneo cells were examined with RT-qPCR and western blot analysis, respectively (Fig. 5A and B). Both VEGFA mRNA and protein levels were significantly reduced in HTR-8/SVneo cells transfected with the miR-203 mimics. By contrast, the expression levels of VEGFA mRNA and protein in the anti-miR-203 group were significantly upregulated in comparison with the control group. Results indicated that VEGFA mRNA expression levels of VEGFA in the different groups was consistent with the protein expression levels. The luciferase reporter assay was conducted to evaluate the direct relationship between the VEGFA 3′-UTR and miR-203. miR-203 overexpression in HTR-8/SVneo cells significantly suppressed the luciferase activity of the wild-type reporter. This inhibition was eliminated when the target miR-203 was mutated (Fig. 5C).