The present study included 20 female patients with EMs who were admitted to the Department of Gynaecology and Obstetrics of the Affiliated Hospital of Jining Medical University (Jining, China) between May 2013 and December 2014. Their eutopic and ectopic endometrium were collected. As a control, normal endometrial tissues were collected from 18 female patients with primary cervical cancers who had undergone complete hysterectomy. The average age of patients with EMs was (43±2.4 years) and that of the control group was (41±3.3 years); there were no significant differences between two groups with regard to age. All patients experienced regular menses and the endometrium was in the secretory phase (confirmed by menstrual cycle and histological examination). Only patients who had not received any hormone treatment and who had not experienced any serious complications in the three months preceding surgery were included in the present study. Prior written and informed consent was obtained from every patient and the study was approved by the ethics review board of the Affiliated Hospital of Jining Medical University (Jining, China).

ESCs were isolated from endometrial tissues. In brief, following rinsing 2–3 times with phosphate-buffered saline (PBS), endometrial tissue was cut into 0.5–1 mm³ sections, which were incubated with collagenase (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37°C for 50–80 min. An equal volume of Dulbecco's modified Eagle's medium (DMEM)/F12 (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) with 10% fetal bovine serum (FBS) (Hyclone; GE Healthcare Life Sciences) was added when no obvious tissue mass was observed. Tissue residues were removed using a 100-mesh filter and the filtrate was centrifuged at 800 × g, for 5 min at 4°C. The pellet was re-suspended in serum-free DMEM/F12 (HyClone; GE Healthcare Life Sciences) and then filtered through a 200-mesh filter. The filtrate was centrifuged at 1,000 × g for 10 min at 4°C and the supernatant was discarded. The cells were suspended in the DMEM/F12 with 10% FBS (containing 100 IU/ml penicillin and 100 IU/ml streptomycin), seeded in culture flasks and incubated at 37°C in 5% CO₂ for 24 h. The DMEM/F12 medium with 10% FBS was replaced and cells were cultured at 37°C in an incubator with 5% CO₂. The medium was changed every 48 h and cell growth was measured every day. Once the cells had reached 80–90% confluence, cells were passaged following a standard procedure.

At 24 h prior to transfection, cells were seeded in 6-well plates at 70–90% confluency. ESCs were divided into four groups based on transfection: An miR-30c mimics group, a miR-30c inhibitor group, a negative control group (NC) and a blank group. These miRs were purchased from Genepharma Co., Ltd. (Shanghai, China). Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used for transfection following the manufacturer's instructions. Cells were harvested 48 h after transfection and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to detect the expression of miR-30c and PAI-1 in the cells.

Total RNA was isolated from tissues using 1 ml TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) per 100 mg tissue according to the manufacturer's protocol. For transfected cells, 2×10⁵ cells were treated with 1 ml TRIzol reagent. RNA integrity was checked by gel electrophoresis and the purity of RNA was assessed using the ratio of absorbance at 260 and 280 nm (Thermo Scientific™ Evolution 300; Thermo Fisher Scientific, Inc.). Complementary (c)DNA was synthesized from total RNA by reverse transcription with the PrimeScript™ RT regent kit (Takara Biotechnology, Inc., Dalian, China) and stored at −20°C. The 20-µl reverse transcription system included 6 µl miRNA template, 2×10 µl miRNA Reaction Buffer mix, 2 µl 0.1% bovine serum albumin (BSA) and 2 µl miRNA PrimeScript RT Enzyme Mixture (all Takara Biotechnology, Inc.). The reaction was performed using ABI Veriti 96-Well Thermo Cycler (Thermo Fisher Scientific, Inc.) at 37°C for 60 min with Poly A primer. The SYBR-Green RT-PCR Master mix (Takara Biotechnology, Inc.) was used to detect the expression of miR-30c and PAI-1 in tissues and ESCs. U6 was used as internal reference for detecting miR-30c, while GAPDH was used for PAI-1. The primer sequences were as follows: U6 forward, GCT TCG GCA GCA CAT ATA CTA AA AT and reverse, CGC TTC ACG AAT TTG CGT GTC AT; GAPDH forward, TTA GCA CCC CTG GCC AAG G and reverse, CTT ACT CCT TGG AGG CCA TG. The primers for miR-30c were 5′-TGTGTAAACATCCTACACTCTCAG-3′ and Uni-miR qRT-PCR Primer (Sangon Biotech Co., Ltd., Shanghai China). The primers for PAI-1 were: Forward, 5′-ACCTGGGAATGACCGACATGT-3′ and reverse, 5′-CTCTCGTTCACCTCGATCTTCACT-3′. The miRNA reaction system included 12.5 µl SYBR RT-PCR Master mix, 1 µl forward primer and 1 µl reverse primer, 2 µl cDNA and 8.5 µl double distilled (dd)H₂O, and the cycling conditions were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 20 sec. The PAI-1 system included 10 µl SYBR RT-PCR Master mix, 0.5 µl forward primer and 0.5 µl reverse primer, 1 µl cDNA and 8 µl ddH₂O. The PCR cycling conditions were as follows: 95°C for 10 min, followed by 40 cycles at 95°C for 1 min, 60°C for 40 sec, 72°C for 30 sec and 72°C for 1 min. Relative expression was calculated using the 2^−ΔΔCq method (32).

For protein isolation, each 50-mg tissue sample was ground into powder with liquid nitrogen and lysed with 600 µl radioimmunoprecipitation assay (50 mM Tris-base, 1 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100 and 1% sodium deoxycholate) lysis buffer. Following centrifugation at the speed of 12,000 × g at 4°C for 5 min, the supernatant was obtained and the protein concentration was assessed using Pierce BCA Protein Assay kit. (Thermo Fisher Scientific, Inc.) A similar method was applied to extract proteins in ESCs. Isolated proteins (10 µl) were separated by 10% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane. The proteins were incubated with primary antibodies for 1 h at room temperature. The primary antibodies were rabbit anti-human PAI-1 (no. 11907; 1:800) and rabbit anti-human GAPDH antibody (no. 2118; 1:2,000). The membranes were then incubated with secondary antibody overnight at 4°C. The secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (no. 7074; 1:1,000). All antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Finally, the membrane was developed by enhanced chemiluminescence plus reagent (EMD Millipore, Billerica, MA, USA). Image Lab™ software (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was applied to analyze the blot images and the intensity of the bands.

The migration assays were performed using Transwell chambers (Corning Inc., New York, NY, USA). The re-suspended ESCs were seeded (1×10⁵ cell/well) in 200 µl serum-free RPMI 1640 medium (Hyclone; GE Healthcare Life Sciences) and placed into the upper chambers, while the bottom chambers contained 750 µl RPMI medium with 10% FBS. After 48 h of culture, cells on the lower side of the membrane were fixed with 4% formaldehyde at room temperature, washed with PBS and stained using crystal violet. Finally, images of the cells were captured under a light microscope at ×200 magnification with five random views and the number of invaded cells was counted.

Four groups of cells transfected with miR-30c mimics, miR-30c inhibitor or negative control as well as blank cells were seeded in 96-well plates (2×10³/well) and 3 replicates were performed for each group. The MTT solution (Beyotime Institute of Biotechnology, Haimen, China) was added to each well at 24, 48, and 72 h. Following incubation at 37°C for 4 h, the absorbance of each well was measured using a BioTek spectrophotometer (Biotek Instruments, Inc., Winooski, VT, USA) at 492 nm and a cell growth curve was constructed.

In each well of a 96-well plate, 50 µl Matrigel (serum-free medium at 1:8 dilution) was added and dried under sterile conditions. Following transfection for 48 h, 2×10⁴ cells/well were cultured in each incubated well. After 60 min of incubation, non-adherent cells were rinsed off and a Cell Counting Kit-8 (Beyotime Company, Jiangsu, China) was used to quantify the attached cells by detection of absorbance at 450 nm using a BioTek spectrophotometer.

The invasion of ESCs was analyzed using Matrigel invasion chambers (growth-factor depleted Matrigel invasion chambers; BD Biosciences, Franklin Lakes, NJ, USA). In the Matrigel chambers, 500 µl serum-free DMEM was added and incubated at room temperature for 1 h to hydrate the Matrigel. Subsequently, 750 µl DMEM containing 20% fetal bovine serum (Hyclone; GE Healthcare Life Sciences) was added to the lower chamber. Successfully transfected cells were collected and re-suspended to 4×10⁵ cells/ml with DMEM containing 0.1% BSA. Following 18 h of incubation at 37°C with 5% CO₂, the cells on the upper side of the membrane were wiped with a cotton swab. The invaded cells on the other side of the chamber were fixed with 4% methanol at room temperature for 10 min. Following staining with 0.1% crystal violet, cells were counted under a microscope.

Values are expressed as the mean ± standard deviation. Statistical analysis was performed using SPSS 16 statistical software (SPSS, Inc., Chicago, IL, USA). All data were analyzed using the Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR was performed to evaluate the expression of PAI-1 and miR-30c in EMs tissues. PAI-1 expression was significantly increased in ectopic and eutopic endometrium compared with normal tissues (P<0.05; Fig. 1A). PAI-I expression did not significantly differ between ectopic and eutopic endometrium. By contrast, the expression of miR-30c was significantly decreased in ectopic and eutopic endometrium compared with normal tissues (P<0.05); however, no difference in miR-30c expression was observed between ectopic and eutopic endometrium (Fig. 1B). In addition, the levels of PAI-1 protein in tissues were detected by western blot analysis and were similar to its mRNA expression. Levels of PAI-1 protein were significantly higher in eutopic and ectopic endometrium than in normal tissues (P<0.05), while there was no significant difference between PAI-1 expression in ectopic and eutopic endometriosis tissue (Fig. 1C). These results demonstrated that miR-30c expression was decreased, whereas PAI-1 expression was increased in EMs tissue.

To determine whether PAI-1 expression is regulated by miR-30c, ESCs were transfected with miR-30c mimics and miR-30c inhibitor. Following transfection for 48 h, miR-30c expression was significantly increased in ESCs transfected with miR-30c mimics and was >70% higher than that in the NC and Blank groups (P<0.05 vs. Blank; Fig. 2A). Furthermore, miR-30c expression was significantly decreased in ESCs transfected with miR-30c inhibitor and was ~30% of that in the NC and Blank groups (P<0.001 vs. Blank; Fig. 2A). Total RNA and protein was extracted from transfected ESCs to detect the expression of PAI-1 mRNA and protein by RT-qPCR and western blotting, respectively. Compared with the NC and Blank groups, cells transfected with miR-30c mimics exhibited significantly decreased expression of PAI-1 at the mRNA (P<0.05; Fig. 2B) and protein level (P<0.05; Fig. 2C). By contrast, inhibition of miR-30c expression increased the expression of PAI-1 at the mRNA and protein level (P<0.05; Fig. 2B and C). These results indicated that overexpression of miR-30c may repress the expression of PAI-1 mRNA and protein.

To further assess the regulatory roles of miR-30c in ESCs, its effect on migration and invasion were assessed. Compared with the Blank and NC groups, ESCs overexpressing miR-30c exhibited significantly reduced cell migration (P<0.05; Fig. 3A) and invasion (P<0.05; Fig. 3B). By contrast, inhibition of miR-30c expression in ESCs increased the ability of cells to migrate and invade (P<0.05; Fig. 3A and B). These results indicated that miR-30c expression inhibits the motility of ESCs.

To elucidate the effect of miR-30c on cell proliferation, an MTT assay was performed. The results demonstrated that, compared with the Blank and NC groups, overexpression of miR-30c in ESCs significantly reduced the proliferation of ESCs (P<0.05), whereas inhibition of miR-30c in ESCs significantly increased ESC proliferation (P<0.05; Fig. 3C). This suggested that miR-30c may repress the proliferation of ESCs.

Compared with the Blank and NC groups, the results of an adhesion test using Matrigel showed that overexpression of miR-30c in the miR-30 mimics group decreased the number of adhesive cells, while the inhibition of miR-30c in the group transfected with the miR-30 inhibitor exhibited an increased number of adhesive cells (Fig. 3D). This indicated that miR-30c may reduce the adhesion ability of ESCs.