The research protocol was approved by the Consultative Committee for the Protection of People in Biomedical Research of Jiangxi Maternal and Child Health Hospital (Nanchang, China). Written informed consent was obtained from each patient prior to participation in the investigation.

Eutopic endometrial stromal cells were derived from the endometrial tissue of 20 female patients (8 proliferative phase and 12 secretory phase cases) with ovarian endometriosis (mean age, 43.6±3.2 years) that presented at the Jiangxi Province Key Laboratory of Molecular Medicine (Nanchang, China). The patients had not received any prior hormone therapy or chemotherapy. A section of each endometrial specimen was submitted for histological diagnosis and the remainder was frozen in liquid nitrogen and later prepared for use in subsequent experiments.

Cells were cultivated in Dulbecco's modified Eagle's medium (DMEM)/F12 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) and incubated at 37°C in a humidified atmosphere containing 5% CO₂. Experimental recombinant lentivirus [small interfering RNA (si)S100A6-enhanced green fluorescent protein (EGFP)-specific] and control recombinant lentivirus (without siS100A6) vectors were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). EGFP was included as a reporter gene. The interference fragment sequence used for S100A6 siRNA was 5′-AAGCTGCAGGATGCTGAAATT-3′, based on previous research findings (17).

Eutopic endometrial stromal cells were seeded in 6-well plates and grown to 60–70% confluence. Cells were then transfected with the control recombinant lentivirus vector (Lv-control), siS100A6 lentivirus vector (Lv-siS100A6), or with no vector (untreated control) using Polybrene^® reagent (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) in accordance with the manufacturer's protocol. All transfections were performed in triplicate. Transfected cells were cultured at 37°C in an atmosphere containing 5% CO₂ for 4 days, and were subsequently used for a range of analytical experiments.

A total of 4 days post-transfection, cells were lysed in ice-cold radioimmunoprecipitation assay buffer containing 1% protease inhibitors (Roche Diagnostics, Basel, Switzerland). Proteins were harvested and the protein concentration was determined using the Bicinchoninic Acid Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Proteins (20 µg) were separated by 8% SDS-PAGE and transferred to a nitrocellulose membrane (EMD Millipore, Billerica, MA, USA). Membranes were blocked for 2 h with 5% non-fat dried milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature, and were then incubated at 4°C overnight with rabbit anti-human β-catenin (1:800; cat. no. ab6302), S100A6 (1:1,000; cat. no. 181974), or β-actin (1:1,000; cat. no. ab6276) primary antibodies (Abcam, Cambridge, UK). Membranes were subsequently washed three times in TBST and incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobin G (1:10,000; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China; cat. no. ZDR-5306) for 2 h at room temperature. Following three washes in TBST, specific bands were detected using the enhanced chemiluminescence system (EMD Millipore). The bands were analyzed by using Gel-Pro Analyzer 4.0 software (Media Cybernetics, Inc., Rockville, MD, USA). The monoclonal anti-β-actin antibody served as an internal control for loading.

A total of 4 days post-transfection, cells were cultivated in 96-well plates at a density of 3×103 cells/well in a total volume of 100 µl/well. A total of 10 µl Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added to each well when the cells reached 40–50% confluence. The absorbance of each well was measured at a wavelength of 450 nm using a microtiter plate reader (Molecular Devices, LLC, Sunnyvale, CA, USA). All experiments were performed in triplicate and results are expressed as the mean ± standard deviation.

Migration assays were conducted in modified Boyden chambers (BD Biosciences, Franklin Lakes, San Jose, CA, USA) with 8-µm pore filter inserts in 24-well plates. A total of 4 days post-transfection, 2×104 cells suspended in serum-free DMEM/F12 were added to the upper chamber. DMEM/F12 supplemented with 20% FBS was added to the lower chamber as a chemoattractant. Following a 48 h cultivation at 37°C, the membrane containing migrated cells was fixed using methanol and stained with crystal violet. Migration was detected by light microscopy following removal of the non-filtered cells on the upper side of the membrane with cotton swabs. Three independent experiments were performed.

A total of 4 days post-transfection, cells were harvested, diluted to a concentration of 3×105 cells/ml, and washed twice with ice-cold phosphate-buffered saline. Subsequently, the cells were incubated in the dark, at room temperature for 15 min with phycoerythrin (PE) Annexin V and 7-aminoactinomycin D (7-AAD) and 380 µl 1X binding buffer from the Annexin V-PE/7-AAD apoptosis kit (Multisciences, Beijing, China) according to the manufacturer's protocol, and were analyzed by flow cytometry. Cells undergoing early apoptosis only bound to PE Annexin V, whereas cells that bound to PE Annexin V and 7-AAD were in the late stages of, or had already undergone, apoptosis. The experiment was repeated three times.

A total of 4 days post-transfection, total RNA was isolated from cells grown to 70–80% confluence using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA (2 µg) was used for the synthesis of first-strand cDNA using First Strand cDNA Synthesis kit (Promega Corporation, Madison, WI, USA). PCR primers were designed using Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA, USA) from reported sequences (Table I). Target gene expression was normalized to β-actin levels.

PCR reactions were performed in a 20-µl volume containing 0.5 µl cDNA, 20 pmol of each primer, 0.25 mM of each dNTP, 1 unit Taq DNA polymerase (Promega Corporation), and the buffer supplied by the manufacturer. β-catenin amplification used the following thermal cycling conditions: Initial denaturation at 94°C for 3 min followed by 35 cycles of denaturation at 94°C for 30 sec, primer annealing at 53°C for 30 sec and primer extension at 72°C for 25 sec, with a final extension at 72°C for 5 min. S100A6 and β-actin amplification was similar, with the exception of an annealing temperature of 57°C. PCR products were separated by 1.5% agarose gel electrophoresis and were subjected to densitometric scanning using an Epson photo scanner (Epson America Inc., Long Beach, CA, USA). Bands were semi-quantified using Gel-Pro Analyzer 4.0 software (Media Cybernetics, Inc., Rockville, MD, USA). All experiments were conducted in triplicate.

Data are presented as the mean ± standard deviation. Statistical analysis was conducted using SPSS software version 19.0 (IBM SPSS, Armonk, NY, USA). Statistical analyses were performed using a one-way analysis of variance and Student's t-test for comparisons between two different groups, or χ2 test. P<0.05 was considered to indicate a statistically significant difference.

Eutopic endometrial stromal cells were transfected with Lv-control or Lv-siS100A6. The efficiency of transfection was assessed 4 days post-transfection by analyzing EGFP expression using fluorescence microscopy, it was indicated that transfection was efficient.

Western blotting and RT-PCR were performed to measure intracellular S100A6 expression 4 days following the transfection of eutopic endometrial stromal cells. S100A6 protein expression was decreased in cells transfected with Lv-siS100A6 compared with untreated and Lv-control-transfected cells, (0.239±0.013, 0.689±0.010 and 0.706±0.008 for Lv-siS100A6 group, untreated group and Lv-control group, respectively; P<0.05), following normalization to β-actin levels (Fig. 1A and B). There was no significant difference in the expression of S100A6 between untreated and Lv-control cells (P>0.05). The mRNA expression levels of S100A6 were also lower in cells transfected with Lv-siS100A6 compared with the untreated and Lv-control-transfected cells (P<0.05; Fig. 1C).

To further examine the interaction between S100A6 and β-catenin, the expression levels of β-catenin were detected using western blotting and RT-PCR, in cells post-transfection with Lv-siS100A6 or Lv-control constructs. As presented in Fig. 1A and D, β-catenin expression was significantly lower in Lv-siS100A6-transfected cells (0.188±0.012) compared with Lv-control (0.794±0.006) and untreated cells (0.787±0.011) following normalization to β-actin levels. In addition, β-catenin mRNA levels were observed to be markedly decreased in Lv-siS100A6-transfected cells, whereas no notable difference was detected in β-catenin levels between Lv-control and untreated cells (Fig. 1E).

To further investigate the functional roles of S100A6 in endometriosis, the present study examined the effects of S100A6 silencing on eutopic endometrial stromal cells. As presented in Fig. 2A, the CCK-8 proliferation assay revealed that the growth rate was significantly reduced in cells transfected with the Lv-siS100A6 construct compared with untreated cells or those transfected with the Lv-control construct (P<0.05).

The effects of siS100A6 on the migratory capacity of eutopic endometrial stromal cells were also investigated. As presented in Fig. 2B and C, the number of migrated cells was significantly reduced in the Lv-siS100A6 group compared with the untreated group or those transfected with Lv-control, (182.00±13.21/per field, 170.00±20.32/per field and 96.00±21.34/per field in untreated, Lv-control-, and Lv-siS100A6-transfected cells, respectively).

To determine whether the siS100A6-induced inhibition of proliferation and migration may be a consequence of the induction of cell death, the present study examined the number of early apoptotic eutopic endometrial stromal cells 4 days post-transfection. Flow cytometric analysis revealed that few Lv-control-transfected (13.73%) or untreated cells (12.25%) underwent early apoptosis, whereas transfection with the Lv-siS100A6 construct significantly increased the percentage of early apoptotic cells to 35.44% (Fig. 3A and B; P<0.05).