The procedures for the collection of the tissue samples were approved by the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University, Urumqi, China. All patients signed an informed consent form in compliance with the code of ethics of the World Medical Association (Declaration of Helsinki). Paired eutopic and ectopic endometrium samples were collected from 20 women with endometriosis who underwent laparoscopic surgical procedures at the First Affiliated Hospital of Xinjiang Medical University. The patients were aged 25 to 42 years, had regular menstrual cycles and had not received hormonal therapy for at least 3 months prior to surgery. A total of 10 samples of endometrium from women with hysteromyoma were used as the controls. The tissues were from the proliferative phase of the menstrual cycle. After collection, the tissue samples were immediately frozen and kept in liquid nitrogen for further analysis.

The CRL-7566 endometriosis cell line was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). This cell line was established from a benign ovarian cyst obtained from a patient with endometriosis. The cells were isolated from both the inner and outer surfaces of the cyst. The cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA). The cells were grown at 37°C in 5% CO₂. The miR-29c mimic, miR-29c inhibitor and negative control (NC), mutant and wild-type c-Jun 3′UTR, as well as the c-Jun overexpression plasmid and the shRNA plasmid were transfected into the cells using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer’s instructions and incubated at 37°C for 24 h.

The targets of miR-29c were predicted by miRanda (http://www.microrna.org/) and c-Jun was identified to be a target of miR-29c. The sequences of human wild-type c-Jun 3′ untranslated region (3′UTR) and mutant c-Jun 3′UTR were then cloned into the pGL3-control luciferase reporter vector (Promega, Madison, WI, USA). c-Jun 3′UTR reporter plasmids were co-transfected with the miR-29c mimic or negative control (NC) into the cells. pRL Renilla luciferase reporter vector was used as the internal control in each assay. Following transfection, cell lysates were collected and luciferase activity was measured using the Dual-Luciferase Reporter assay system (Promega).

MTT assay was used to evaluate cell proliferation. The cells were seeded into 96-well culture plates at a density of 1×10⁴ cells/well. Following transfection, 10 μl MTT solution (0.5 mg/ml; Beyotime, Shanghai, China) were added to each well followed by incubation for 4 h. After dissolving the MTT formazan with DMSO (Sigma, St. Louis, MO, USA), cell proliferation was determined by reading the plates at 570 nm on a Multiskan Ascent 354 microplate reader (Thermo Labsystems, Waltham, MA, USA).

Cell invasion assay was performed using Matrigel-coated Transwell plates (Corning, New York, NY, USA). Cells in serum-free medium at a final density of 5×10⁴ cells/ml were added to the upper chambers and incubated at 37°C for 12 h, and cell medium containing 10% FBS was added to the lower chambers. Following incubation, non-invaded cells were removed using cotton swabs, and the invaded cells were fixed in 95% ethanol and stained with hematoxylin. The number of invaded cells was determined by counting the stained cells under an inverted microscope (TS100; Nikon, Tokyo, Japan).

Cell apoptosis was assessed by FCM. After washing with phosphate-buffered saline (PBS), the cells were resuspended in binding buffer and stained with Annexin V and propidium iodide (PI) (Kaiji Biological Inc., Nanjing, China) following the manufacturer’s instructions. Positive cells were detected and quantified by FCM (BD FACSAria; BD Biosciences, Franklin Lakes, NJ, USA).

Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The miRNAs were isolated using a miRNeasy Mini kit (Qiagen, Hilden, Germany). Reverse transcription was performed using a RevertAid™ First Strand cDNA Synthesis kit (Fermentas, Vilnius, Lithuania) and quantitative (real-time) PCR was carried out on a 7900 Real-Time PCR System using a SYBR-Green PCR kit (both from Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. The results were analyzed using the comparative method following normalization of the expression values to U6.

Protein samples were extracted using a total protein extraction kit (Kaiji Biological, Inc.) and the protein concentration was determined using the BCA kit (Beyotime). The samples were separated in 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% non-fat milk overnight at 4°C. After washing with PBS, the membranes were incubated with primary antibody [mouse monoclonal to c-Jun (ab119944), mouse monoclonal to β-actin (ab8226); Abcam, Cambridge, MA, USA] overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibody (BA1050; Boster, Wuhan, Hubei, China) for 1 h at room temperature. The ECL detection kit (Pierce, Rockford, IL, USA) was used to detect the proteins.

Data were presented as the means ± standard deviation (SD). The two-tailed Student’s t-test was used to statistically analyze data between 2 groups. A P-value <0.05 was considered to indicate a statistically significant difference.

The differential expression of miR-29a, miR-29b, miR-29c in the endometrium samples from women without endometriosis, as well as in the eutopic and ectopic endometrium samples was determined by RT-qPCR. The results revealed that the expression of miR-29c progressively decreased from the samples of endometriosis to the those of the eutopic and ectopic endometrium, and the expression of miR-29c between the eutopic and ectopic endometrium was differed significantly (P<0.01). However, the expression levels of miR-29a and miR-29b did not differ significantly between the 3 groups (Fig. 1).

The expression of c-Jun in the endometrium samples from women without endometriosis, as well as in the eutopic and ectopic endometrium samples was examined by western blot analysis. There was no statistically significant difference in c-Jun expression between the group without endometriosis and the eutopic group (Fig. 2). However, the protein expression level of c-Jun was significantly higher in the ectopic group compared with the eutopic group (P<0.01).

In order to determine whether c-Jun is a direct target of miR-29c, wild-type c-Jun-3′UTR or mutant c-Jun-3′UTR were co-transfected with the miR-29c mimic or NC. Luciferase assay revealed that transfection with the miR-29c mimic significantly inhibited the luciferase activity of wild-type c-Jun-3′UTR (P<0.01); however, the luciferase activity of mutant c-Jun-3′UTR did not differ statistically following transfection with the miR-29c mimic or NC (Fig. 3).

Furthermore, the effect of miR-29c on c-Jun expression was examined by western blot analysis. The results from RT-qPCR revealed that miR-29c expression was significantly upregulated in the miR-29c mimic-transfected group and downregulated in the miR-29c inhibitor-transfected group (Fig. 4A). Western blot results revealed a significantly decreased expression of c-Jun in the cells transfected with the miR-29c mimic; however, the expression of c-Jun was increased in the cells transfected with the miR-29c inhibitor (Fig. 4B).

The open reading frame (ORF) clone of homo c-Jun was subcloned into the pcDNA3.1 vector to induce the overexpression of c-Jun. We suppressed c-Jun expression using a specific shRNA plasmid. Transfection with the c-Jun overexpression plasmid did not alter the increase in miR-29c expression in the miR-29c mimic-tansfected cells (Fig. 5A). In addition, the c-Jun shRNA plasmid did not alter the decrease in miR-29c expression in miR-29c inhibitor-tansfected cells (Fig. 5B).

Cells transfected with the miR-29c mimic and c-Jun over-expression plasmid showed a significantly increased protein expression of c-Jun compared with the cells transfected with the miR-29c mimic only (Fig. 6A). However, transfection with c-Jun shRNA significantly downregulated the expression of c-Jun in the cells transfected with the miR-29c inhibitor (Fig. 6B).

In order to examine the effects of miR-29c on cell proliferation, the miR-29c mimic or inhibitor were transfected into the cells, and MTT assay was used to analyze cell proliferation. Compared with the NC-transfected cells, the miR-29c mimic-transfected cells proliferated at a significantly lower rate (Fig. 7A). By contrast, transfection with the miR-29c inhibitor promoted cell growth (Fig. 7B).

Furthermore, the overexpression of c-Jun reversed the inhibitory effects of the miR-29c mimic on cell proliferation (Fig. 7A). Consistent with these results, transfection with c-Jun shRNA reversed the inductive effects of the miR-29c inhibitor on cell proliferation (Fig. 7B).

Cell invasion ability was determined by Transwell-Matrigel assay. Transfection with the miR-29c mimic resulted in significantly decreased cell invasion in comparison to transfection with NC; however this effect was reversed by transfection with the c-Jun overexpression plasmid (Fig. 8A). As expected, transfection with the miR-29c inhibitor significantly increased cell invasion ability, which was reversed by transfection with c-Jun shRNA (Fig. 8B).

FCM was used to examine the cell apoptotic rates. The results revealed that compared with the cells transfected with NC, the miR-29c mimic-transfected cells showed an increased cell apoptotic rate, while transfection with miR-29c inhibitor had the opposite effect. The overexpression of c-Jun reversed the increase in the cell apoptotic rate induced by the transfection with the miR-29c mimic. Consistent with these results, transfection with c-Jun shRNA reversed the inhibitory effects of the miR-29c inhibitor on cell apoptosis (Fig. 9).