For the identification of PIH-specific serum miRNAs, a total of 40 patients were selected: 20 healthy female volunteers as controls and 20 female patients with PIH, were recruited from the Third Affiliated Hospital, Sun Yat-sen University (Guangzhou, China; Table I) between December 2014 and March 2015, having given informed consent to be included in the present study. Differentially expressed miRNAs were directly validated using RT-qPCR according to previous research (19). As presented in Table I, the exclusion criteria for both groups included: Patients with kidney disease or essential hypertension, a history of alcohol or drug abuse, and illegal drug addiction within the 6 months prior to signing the informed consent. Furthermore, the PIH patients were all pathologically diagnosed by doctors and all blood samples were collected prior to any surgery, chemotherapy and/or radiation treatment.

From each patient, 5 ml venous blood was collected on first admission to the hospital. Blood was drawn into a sterile tube without anticoagulant to harvest cell-free serum. The tube was left in a standing position for 20 min prior to centrifugation at 20°C and 1,500 × g for 10 min. The supernatant serum was quickly removed by pipette and stored immediately at −80°C until analysis. The present study was approved by the Ethics Committee from the Third Affiliated Hospital, Sun Yat-sen University and a signed informed consent form was obtained from each participant prior to the study.

The human choriocarcinoma (JAR) cell line was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). JAR cells were cultured for 24 h in growth media containing high glucose-Dulbecco's modified Eagle's medium and supplemented with 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) and 1% penicillin/streptomycin (Mediatech, Inc., Manassas, VA, USA) in a humidified atmosphere of 5% CO₂ and a temperature of 37°C.

Following dilution into single cell suspensions and seeding into 96-well plates (1×10⁴ cells/well), a JAR cell hypoxic model was induced using an AnaeroPack^® system (Mitsubushi Gas Chemical America, Inc., New York, NY, USA) for 48 and 72 h, respectively, prior to harvesting for total RNA isolation (20).

TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to prepare total RNA and subsequently, 75% ethanol replaced isopropanol for RNA precipitation, according to the manufacturer's protocol. RNA quality was determined using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Inc., Wilmington, DE, USA). A total of 1 µg RNA was reverse-transcribed into cDNA using a DBI Bestar^® qPCR RT kit (DBI Bioscience, Ludwigshafen, Germany) according to the manufacturer's protocol.

RT-qPCR was performed using a 7500 Fast Real-Time PCR System Light Cycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). The 20 µl PCR reaction included 1 µl reverse transcription product (1:5), 0.5 µl sense primer, 0.5 µl universal reverse primer and 10 µl DBI-2043 Bestar^® Real time PCR Master Mix (DBI Bioscience). The reactions were incubated at 94°C for 2 min in a 96-well optical plate, followed by 40 cycles of 94°C for 20 sec, 8°C for 20 sec and 72°C for 20 sec. All reactions were completed in triplicate and primer sequences are listed in Table II. mRNAs were quantified using the 2^−ΔΔCq formula (21).

miR-204-5p inhibitor (Shanghai GenePharma, Ltd., Shanghai, China) was transfected into the hypoxic JAR cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) prior to incubation in DMEM without FBS at 37°C for 48 h. JAR cells were then transfected with 100 nM miR-204-5p inhibitor and subjected to the hypoxia precondition for 48, 72 and 96 h respectively. Then, 100 µl Cell Counting Kit-8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added to each well and incubation was completed for 1 h at 37°C. Absorbance was measured at 450 nm using a microplate reader.

Following transfection of the miRNA inhibitor in the hypoxic environment for 48 h, quantification of the apoptotic cells was completed using the Annexin V-FITC/PI apoptosis detection kit (Merck Millipore, Darmstadt, Germany). JAR cells were collected by trypsin digestion method (22), washed with phosphate buffered saline (PBS) and re-suspended in 200 µl binding buffer containing 5 µl Annexin V (10 µg/ml) in DMEM with FBS at 37°C for 10 min in the dark. The cells then underwent incubation with 10 µl PI (20 µg/ml) for 15 min and samples were analyzed using an EPICS^® XL™ flow cytometer (Beckman Coulter, Inc., Brea, CA, USA). Data acquisition and analysis were performed using CellQuest™ software version 5.1 (BD Biosciences, Franklin Lakes, NJ, USA) (23,24).

Following transfection of the miRNA inhibitor into the hypoxic environment and incubation at 37°C for 48 h, JAR cells were collected by trypsin digestion method and washed with PBS prior to re-suspension in 250 µl DMEM. Cold (4°C) dehydrated ethanol (99%) was added to this buffer and incubated overnight at 4°C. Following treatment, cells were collected and incubated with 200 µl PI (20 µg/ml) using a cell cycle assay kit (Vazyme Biotech, Co., Ltd., Nanjing, China) at 37°C for 15 min. Samples were immediately analyzed using flow cytometry (EPICS^® XL™; Beckman Coulter, Inc.). Data acquisition and analysis were performed using CellQuest software version 5.1 (BD Biosciences) (23,24).

For RT-qPCR data analysis, the relative quantification method was used to determine the changes in the expression of the target miRNAs. U6 RNA was used to normalize the expression and change in amplification. The fold change in expression was calculated for each sample using 2^−ΔΔCq, where ΔΔCq=(Cq target gene-CqU6) PIH-(Cq target gene-CqU6) control (25). A value of 2^−ΔΔCq >1.5 or <0.67 was considered to represent differentially expressed miRNA. The Welch t-test was used to assess the differential expression of miRNA measured by RT-qPCR.

For other data analysis, Statistical analysis was performed using SPSS, version 17.0 (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance was used to compare log₁₀-transformed relative quantities of target miRNAs between all groups. Bartlett's test was used to assess the differences in variance between genes. P<0.05 was considered to represent a statistically significant difference for all experiments.

The expression profile of eight serum microRNAs were selected, based on a previous study, (under review, data not shown), for further examination in 18 PIH patients that represented a significant 2-fold change using RT-qPCR. Expression levels of miR-197-3p, miR-92b-3p, miR-342-3p, miR-26a-5p, miR-198, miR-204-5p, miR-296-5p and miR-95-5p were measured in the serum samples of 12 patients with PIH and 6 normal controls (Fig. 1). The results showed significantly elevated expression of miR-197-3p, miR-92b-3p, miR-26a-5p, miR-198 and miR-204-5p in patients with PIH compared with controls (P<0.001; Fig. 1A, B and D-F). However, levels of miR-342-3p, miR-296-5p and miR-95-5p not differ significantly between PIH patients and controls (Fig. 1C and G-H).

The expression of miR-197-3p, miR-92b-3p, miR-26a-5p, miR-198 and miR-204-5p were selected to be assessed in vitro, on the basis of the results of the aforementioned clinical sample examination. Only miR-204-5p expression increased significantly in a time-dependent manner in hypoxic JAR cells (0, 48 and 72 h; Fig. 2).

The effect of miR-204-5p inhibitor on JAR cell proliferation was subsequently assessed. In hypoxia pre-treatment JAR cells, cellular proliferation was enhanced significantly in a time-independent manner in the miR-204-5p inhibitor group, as indicated by the number of cells detected at each time point (P<0.001; Fig. 3).

Apoptosis and cell cycle distribution were analyzed using flow cytometry following the transfection of miR-204-5p inhibitor and hypoxia pre-treatment for 48 h. Compared with the control group, JAR cell exposure to miR-204-5p inhibitor exhibited typical protection from apoptotic morphology with nuclear fragmentation, cell shrinkage and cellular rupture into debris. Apoptosis occurred at a significantly higher rate in cells treated with the negative control compared with the group treated with miR-204-5p inhibitor (P<0.01; Fig. 4). Assessment of the cell cycle indicated that the ratio of cells in the G2/G1 phase increased, although this increase was not significant. However, G1 cell cycle arrest was significantly reduced following transfection with the miR-204-5p inhibitor (P<0.05; Fig. 5).