Female Sprague-Dawley rats (6–7 weeks old; 180–200 g; n=12) were housed at 22–24°C, 55–60% humidity with a 12-h/12-h light/dark cycle, and were given a regular chow diet and water ad libitum. The present study was approved by the Animal Care and Use Committee of Affiliated Qilu Hospital of Shandong University (Jinan, China). All rats were randomly assigned to control (n=6) and Ems (n=6) groups. In the control group, rats were anesthetized with 35 mg/kg pentobarbital sodium without intervention. Rats were anesthetized with 35 mg/kg pentobarbital sodium, and a vertical incision in the abdomen was made. The uterus was removed and immediately washed with PBS, the endometrium was cut into 0.5×0.5-cm sections and uterine segments were sutured onto the peritoneum close to blood vessels. 0.5 µg/kg/day of Estradiol benzoate (MedChem Express, Shanghai, China) was subcutaneously injected for 3 days following the surgery. Following treatment with Estradiol benzoate for 3 days, rats were sacrificed using decollation under 35 mg/kg pentobarbital sodium.

Endometrial stromal cells were separated from the isolated endometrial tissues, and tissue was finely minced. Cells were dispersed and incubated in Dulbecco's modified Eagle's medium (DMEM)/F-12 (Gibco; Thermo Fisher Scientific, Inc.), penicillin-streptomycin solution, and 2 mg/ml collagenase II for 1 h at 37°C. Subsequently, endometrial stromal cells were separated using a 100 µm filter. Stromal cells were pelleted by centrifugation at 200 × g for 10 min at 4°C and incubated with DMEM/F-12 containing fetal bovine serum (10%, v/v; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37°C in a humidified atmosphere containing 5% CO₂. Then, transfection with 100 ng of miR-33b (5′-GUGCAUUGCUGUUGCAUUGC-3′ and 5′-AAUGCAACAGCAAUGCACUU-3′), anti-miR-33b (forward, 5′-CCAAGGATCTCCAGGCTCGAA-3′ and reverse, 5′-TTCGAGCCTGGAGATCCTTGG-3′), small interfering (si)-ZEB1 (sc-38643; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and negative mimics (forward, 5′-TTCTCCGAACGTGTCACGT-3′ and reverse, 5′-ACGTGACACGTTCGGAGAA-3′) was performed using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. Following transfection for 4 h, old medium was removed and new DMEM/F-12 was added into cell. Next, transfection of miR-33b or negative mimics was performed for 4 h. For Wnt activation, 10 µM of SKL2001 (MedChemExpress, Shanghai, China) was added to the cells for 72 h.

Total RNA was extracted from frozen tissues or cells using TRIzol^® reagent (Life Technologies; Thermo Fisher Scientific, Inc.). Total RNA (1 µg) was transcribed to first-strand cDNA using an RT kit (Toyobo Life Science, Osaka, Japan). The RT-qPCR protocol was performed using SYBR-Green PCR master mix (Bio-Rad Laboratories, Hercules, CA, USA) and an Applied Biosystems 7300 Real-Time PCR System (Thermo Fisher Scientific, Inc.). The primer sequences were: miR-33b forward, 5′-ATTCTTTCGAACTGTCTTGG-3′ and reverse, 5′-TCACCCTCGGCTGTCCTGACA-3′; U6 forward, 5′-AGTACCAGTCTGTTGCTGG-3′ and reverse, 5′-TAATAGACCCGGATGTCTGGT-3′. The qPCR cycle was set to an initial 95°C for 10 min, followed by 40 cycles of 95°C for 25 sec, 60°C for 30 sec, and 72°C for 30 sec. Gene expression levels were measured using the 2^−∆∆Cq method (13).

Isolated RNA was reverse transcribed into cDNA and hybridized to Affymetrix HG-U133 Plus 2.0 GeneChip arrays l (Affymetrix, Santa Clara, CA, USA). Data were analyzed through TargetScan version 7.1 (http://www.targetscan.org) and QIAGEN's Ingenuity Pathway Analysis (IPA, QIAGEN, Redwood City, USA) (14).

Endometriosis tissue was collected and fixed with 4% paraformaldehyde for 24 h. The colonic sections of 4 µm were cut from formalin-fixed, paraffin-embedded tissue blocks. Tissue samples were stained with HE assay for 15 min and evaluated under the light microscope (Olympus BX51).

MTT (5 mg/ml; Sigma Aldrich; Merck KGaA) was added to the cells for incubation at 37°C for 4 h. Subsequently, the old medium was removed and dimethyl sulfoxide was added to the cells for 20 min at 37°C. The absorbance was measured by spectrophotometry with a microplate reader (model 680; Bio-Rad Laboratories, Inc.) at 490 nm. Endometrial stromal cells were washed with PBS and resuspended in 100 µl 1X binding buffer (BestBio, Shanghai, China) and incubated with Annexin V-fluorescein isothiocyanate (FITC; BestBio) and propidium iodide (PI; BestBio) for 15 min at room temperature in the dark. The apoptosis rate was analyzed using a flow cytometer (C6; Beckman Coulter Inc., Brea, CA, USA) analyzed using Image Lab version 3.0 (Bio-Rad Laboratories, Inc.).

LDH activity levels were measured using LDH activity kits (C0016; Beyotime Institute of Biotechnology, Haimen, China). The absorbance was measured by spectrophotometry with a microplate reader (model 680; Bio-Rad Laboratories, Inc.) at 450 nm.

Endometrial stromal cells were lysed with lysis buffer (radioimmunoprecipitation assay buffer) containing a protease inhibitor cocktail (phenylmethanesulfonyl fluoride) and EDTA at 4°C for 30 min. Cells were centrifuged at 12,000 × g for 10 min at 4°C. Subsequently, the concentration of total protein was determined using a bicinchoninic acid (BCA) assay, and 10 µg/lane total protein was used to analyze caspase 3/9 activity levels using caspase 3/9 activity kits (C1115 and C1158, Beyotime Institute of Biotechnology). The absorbance was measured by spectrophotometry with a microplate reader (model 680; Bio-Rad Laboratories) at 405 nm.

Endometrial stromal cells were lysed with lysis buffer (radioimmunoprecipitation assay buffer) containing protease inhibitor cocktail (phenylmethanesulfonyl fluoride) and EDTA at 4°C for 30 min. Cells were centrifuged at 12,000 × g for 10 min at 4°C. Subsequently, the concentration of total protein was determined using the BCA assay, and 40 µg/lane total protein was subjected to 10–12% SDS-PAGE and subsequently transferred onto a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc.). The membrane was blocked with 5% non-fat milk in TBS containing 0.1% Tween-20 for 1 h at 37°C, and subsequently incubated with antibodies against Wnt (sc-376029; 1:1,000), β-catenin (sc-65480; 1:1,000), ZEB1 (sc-515797; 1:1,000), apoptosis regulator BAX (Bax; sc-20067; 1:1;000) and GAPDH (sc-51631; 1:5,000; all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C. Following this, membranes were incubated with anti-rabbit immunoglobulin G secondary antibody (sc-2004; 1:5,000; Santa Cruz Biotechnology, Inc.) at 37°C for 1 h and were developed using an enhanced chemiluminescence kit (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Protein levels were quantified using Bio-Rad Laboratories, Inc. Quantity One software (version 3.0).

All data are expressed as the mean ± standard error using SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA). Statistical differences were measured using one way analysis of variance with Bonferroni's correction for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

Firstly, H&E staining suggested that Ems was successfully induced in the Ems group compared with the control group (Fig. 1A). As demonstrated in Fig. 1B and C, miR-33b expression was upregulated by 4.29±0.05 fold in the Ems rat model compared with the control group.

Notably, miR-33b expression was investigated using miR-33b mimics or anti miR-33b mimics in vitro. Fig. 2A and B indicate that miR-33b expression was upregulated by 5.26±0.04 fold or downregulated by 0.26±0.03 fold in the in vitro Ems model compared with the control group. Overexpression of miR-33b suppressed cell viability by 0.82±0.06 fold (48 h) or 0.51±0.06 fold (72 h), and enhanced the lactate dehydrogenase (LDH) activity of Ems by 4.70±0.29 fold; additionally, downregulation of miR-33b promoted cell viability by 1.23±0.06 fold (48 h) or 1.28±0.03 fold (72 h), and decreased the LDH activity of Ems by 0.37±0.03 fold (Fig. 2C-F).

Subsequently, it was demonstrated that overexpression of miR-33b increased the apoptosis rate by 2.05±0.15-fold and promoted caspase-3 and 9 activity by 3.97±0.17 and 3.70±0.24-fold, respectively, in the Ems model compared with the control group (Fig. 3A-D). However, downregulation of miR-33b decreased the apoptosis rate by 0.54±0.04-fold (Fig. 3E and F) and the caspase-3 and 9 activity by 0.39 ± 0.03 and 0.31 ± 0.05-fold, respectively, in the Ems model (Fig. 3G and H).

To analyze the mechanism of miR-33b in Ems, the Wnt/β-catenin signaling pathway was investigated. It was demonstrated that miR-33b was able to target the 3′UTR of ZEB1 (Fig. 4A). Subsequently, overexpression of miR-33b suppressed ZEB1 protein expression by 0.34±0.04 fold, reduced Wnt and β-catenin protein expression by 0.44±0.06 and 0.32±0.12 fold, respectively, and induced Bax protein expression by 2.56±0.13 fold in the in vitro Ems model (Fig. 4B-F). Furthermore, downregulation of miR-33b induced ZEB1 protein expression by 1.94±0.17 fold, reduced Wnt/β-catenin protein expression by 2.57±0.07 and 2.72±0.14 fold, respectively, and suppressed Bax protein expression by 0.43±0.02 fold in the in vitro Ems model compared with the control group (Fig. 4G-K).

To examine the role of ZEB1/Wnt/β-catenin in the effect of miR-33b on Ems, si-ZEB1 or Wnt agonist were co-transfected with miR-33b mimics in Ems. As illustrated in Fig. 5A-E, compared with the miR-33b downregulation group, si-ZEB1 suppressed ZEB1 by 0.52±0.08 fold, decreased Wnt and β-catenin protein expression by 0.57±0.04 and 0.71±0.05 fold, respectively, and induced Bax protein expression by 2.36±0.07 fold in the Ems model. Furthermore, compared with the miR-33b overexpression group, the Wnt agonist induced Wnt and β-catenin protein expression by 2.01±0.06 and 1.61±0.05 fold, respectively, and suppressed Bax protein expression by 0.61±0.07-fold in the in vitro Ems model compared with miR-33b (Fig. 5F-I).

To gain insight into the function of miR-33b on Ems, the effect of the ZEB1 inhibitor on the function of miR-33b in Ems was analyzed. Compared with the miR-33b downregulation group, ZEB1 inhibition reduced cell viability by 0.81±0.05 (48 h) or 0.71±0.08-fold (72 h), increased LDH activity by 2.12±0.04 fold, increased caspase-3 and 9 activity by 2.72±0.14 and 2.16±0.12-fold, respectively, and apoptosis rate by 2.26±0.07 fold in the in vitro Ems model (Fig. 6).

Furthermore, miR-33b mimics were co-transfected with Wnt agonist in the in vitro Ems model. Compared with the miR-33b overexpression group, Wnt activation inhibited cell growth by 1.46±0.06 (48 h) or 1.86 ± 0.05 fold (72 h), LDH activity by 0.36±0.13 fold, caspase-3 and 9 activity by 0.39±0.03 and 0.48±0.12 fold, respectively, and apoptosis rate by 0.61±0.07 fold in the in vitro Ems model following miR-33b (Fig. 7).