Progesterone receptor antagonist provides palliative effects for uterine leiomyoma through a Bcl-2/Beclin1-dependent mechanism

Uterine leiomyoma is the most common benign smooth muscle tumor of uterus in women of reproductive age, with a high lifetime incidence. Nowadays, the exploration on the pharmacotherapies, such as progesterone receptor antagonist (PRA) requires more attention. Hence, the current study aimed to examine whether mifepristone, a PRA, influences the autophagy and apoptosis of uterine leiomyoma cells. Primary uterine leiomyoma cells were collected from 36 patients diagnosed with uterine leiomyoma to establish PR-M-positive (PR-M[+]) cells. The lentiviral vector overexpressing or silencing PR-M was subsequently delivered into one part of PR-M(+) cells in order to evaluate the role of PR-M in PR-M(+) cells. The results obtained revealed that cell viability was increased, while cell autophagy and apoptosis were diminished in the PR-M(+) cells treated with overexpressed PR-M, whereby the Bcl-2 level was elevated and the level of Beclin1 was reduced. An opposite trends were identified following treatment with knockdown of PR-M. Mifepristone at different concentrations (low, moderate, or high) was then applied to treat another part of the PR-M(+) cells. Mifepristone was identified to promote cell autophagy and apoptosis, decrease Bcl-2 level and increase Beclin1 level, accompanied by weakened interaction between Bcl-2 and Beclin1. Moreover, these effects of mifepristone on PR-M(+) cells were enhanced with increasing of the concentration. Taken together, the present study present evidence indicates the ability of PRA to regulate the Bcl-2/Beclin1 axis, ultimately promoting the autophagy and apoptosis of uterine leiomyoma cells, highlighting that PRA serves as a promising therapeutic target for the treatment of uterine leiomyoma.


Introduction
Uterine leiomyoma, also commonly referred to as uterine fibroids, remains the most common type of benign tumor afflicting the smooth muscle of the uterus, with the lifetime prevalence exceeding approximately 70% in the general population [1]. Uterine leiomyoma is characterized by heavy or prolonged menstrual bleeding and resultant anemia generally affecting women of reproductive age, which may significant affect the quality of life and fertility [2]. At present, surgeries (e.g. hysterectomy and myomectomy by hysteroscopy or laparoscopy) and pharmacotherapies (e.g. oral contraceptives, progestins, and GnRH analogs) represent the first-line therapeutic approaches for patients diagnosed with uterine leiomyoma [3,4]. Emerging evidence has demonstrated that progesterone and its receptors (PRs) may act to promote the uterine leiomyomata cell viability [5]. Although surgical intervention has been reported as an effective measure, it is an approach that is financially damaging for most patients [3]. Therefore, in order to find an alternative measure to treat uterine leiomyoma and reduce the need for invasive surgery, treatments with anti-progestins and selective PR modulators (SPRMs), like progesterone receptor antagonist (PRA), require further investigation. As a steroid hormone, progesterone represents a crucial element involved in the coordination of the reproductive physiology of normal mammalian female, which is predominately secreted by the corpus luteum developed in the ovulated ovary. Ovarian progesterone represents an essential regulator of uterine function, which elicits effects on numerous tissues and organs, including the breast uterus, as well as the ovary [6]. Evidence has been presented indicating that volume maintenance and growth of uterine leiomyoma are progesterone dependent events [7]. The action of progesterone on the female reproductive system is largely mediated via PR which is synthesized from a single gene and expressed as two main protein isoforms (PR-A and PR-B) [8]. Moreover, PRs, involving in the actions of the ovarian steroid progesterone, play a critical role in gonadotropin secretion, endometrium implantation, pregnancy maintenance, and breast tissue differentiation [9]. One truncated progesterone receptor (PR-M) only encodes exons 4 with 16 unique N-terminal amino acids and is involved in some diseases [10]. Two classical isoform of progesterone receptors, including PR-A and B, are integrate pattern, in which isoform A represents ligand-dependent transdominant repressor, while isoform B represents transcriptional activator of some hormone-related promoters [11,12]. However, little evidence determines the short receptor, PR-M, exhibit similar physiological function of PR-A or PR-B. Recent studies have demonstrated that PR-M localizes to outer membrane of mitochondrion and is a regulatory participant in cellular respiration [10]. A progesterone-dependent elevation in cellular respiration via PR-M is presented. Furthermore, a large number of studies have highlighted the role of progesterone and PR in various types of cancers [13]. PR-M has been identified in breast cancer cells and has a progesterone-dependent effect on proliferation of cancer cell [10,14]. In addition to its involvement in cancer diseases, a recent investigation indicated that PR-M is strongly associated with heart failure in transgenic mouse models. The aforementioned literature suggests that PR-M is a metabolic regulator capable of regulating cellular respiration. The role of SPRMs in the treatment of uterine leiomyoma has been evidenced in previous reports, highlighting SPRMs as an integrator of proliferation in uterine leiomyoma cells with potential benefit to those seeking to preserve future fertility [15,16]. Based on the Clinical Trial database (http://clinicaltrials.gov) (the US National Institutes of Health), several treatment options are under clinical trial, including mifepristone (MIF), asoprisnil, ulipristal, aromatase inhibitors, epigallocatechin gallate, and pirfenidone [17]. A prospective randomized placebo controlled trial has reported that MIF, a PRA, may offer an effective therapeutic option for women with uterine leiomyoma [18]. Recent reports have implicated PRs in the development of certain cancers via the modulation of the apoptosis-related factor Bcl-2 or autophagy-related factor Beclin1 [19][20][21]. In the current study, we employ MIF at varying concentrations to treat primary uterine leiomyoma cells isolated from patients diagnosed with uterine leiomyoma, in order to determine whether PRA could be a therapeutic option for the treatment of uterine leiomyoma. Hence, we examined the hypothesis that the underlying mechanism may be related to the Bcl-2/Beclin1 axis.

Study subjects
A total of 36 patients diagnosed with uterine leiomyoma, who had previously undergone a myomectomy by laparotomy at the Third Affiliated Hospital of Zhengzhou University (Zhengzhou, China) from July 2016 to January 2018 were enrolled for the present study. The enrolled patients were aged from 31 to 50 years old, with a mean age of 40.56 + − 6.41 years. All enrolled patients were confirmed not to be suffering from other hormone related diseases and had not received any steroid hormone medications at least 6 months before surgery. All specimens were diagnosed by pathological examination. The present study was conducted with the approval of the Ethics Committee of the Third Affiliated Hospital of Zhengzhou University (Zhengzhou, China) and in strict adherence with the Helsinki Declaration. Written informed consent was obtained from each patient prior to enrollment and collection of their respective primary uterine leiomyoma cells as well as endometrium biopsy samples.

Selection of the primary PR-M-positive uterine leiomyoma cells
The collected specimens were rinsed with phosphate buffer solution (PBS) containing penicillin/streptomycin for 3-5 min, and then treated with 0.2% type I collagen at 37 • C for 2-3 h. The solution was then filtered, centrifuged at 1000 r/min for 10 min with supernatant discarded, followed by incubation with 10% complete medium in 5% CO 2 at 37 • C for 24 h. The cells observed to have adhered were passaged using 0.25% trypsin-EDTA solution, when cell confluence reached 80%. Cellular morphology was analyzed under an inverted phase contrast microscope (DM500/DM750, Leica, Wetzlar, Germany). The cells were subsequently identified by immunohistochemistry means. The cells at the third passage were then inoculated into six-well plates. When cells reached 80-90% confluence, the total protein was extracted and analyzed by Western blot analysis in order to identify the PR-M-positive (PR-M

Immunohistochemistry
The PR-M(+) cells were trypsinized and inoculated in six-well plates. Upon reaching 70-80% confluence, the cells were fixed in 4% paraformaldehyde for 30 min and blocked using normal goat serum for 1 h at room temperature. After the removal of goat serum, the cells were incubated with primary antibody anti-smooth muscle Actin

Co-immunoprecipitation
The PR-M(+) cells were lysed in a lysis buffer containing proteinase (Hoffmann-La Roche Ltd, Basel, Switzerland) and phosphatase (Thermo Scientific, Inc., Waltham, Massachusetts, U.S.A.) inhibitor at 4 • C for 30 min. After the cell lysates had been centrifuged at 16000 g at 4 • C for 30 min, the supernatant was pre-treated with 60 μl protein G agarose for 2 h and incubated overnight with Bcl-2-agarose (1:1000, sc-7382, Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), or with IgG (1:5000, ab6789, Abcam). The mixture was then re-suspended with 2 × sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) loading buffer and boiled for 5 min. Finally, the protein level of Beclin1 was determined by Western blot analysis.

Western blot analysis
The PR-M(+) cells were lysed in a lysis buffer containing proteinase and phosphatase inhibitor at 4 • C for 30 min and centrifuged at 10,000 r/min for 15 min. The cell lysates were then separated using 10% PAGE followed by transfer onto a polyvinylidene fluoride (PVDF) membrane. Membrane was blocked using 5% skim milk for 1 h at room temperature, followed by incubation at 4

Monodansylcadaverine staining for cell autophagy
The PR-M(+) cells were incubated on coverslips coated with polylysine. The autophagosomes were subsequently labeled with 20 μM MDC for 30 min and analyzed under a fluorescence microscope. The fluorescence of MDC was observed to be in a punctate distribution within the cytoplasm when the autophagosome was formed. Image J software was applied for quantitative fluorescence analysis.

Cell counting kit-8 assay for cell viability
The PR-M(+) cells were treated with 0.25% trypsin and constructed into the single cell suspension with the density adjusted to 5 × 10 4 cells/ml. The cell suspension was then inoculated into 96-well plates (100 μl/well) and cultured in 5% CO 2 at 37 • C for 24 and 48 h, respectively, with five duplicated wells set for each group. After a 24-or 48-h incubation, each well was incubated with 100 μl of CCK-8 reagent (Beyotime Biotechnology, Shanghai, China) for a

Flow cytometry
The PR-M(+) cells were treated with 0.5 ml of 0.25% trypsin and subsequently constructed into a cell suspension with the density adjusted to 1 × 10 6 cells/ml. The cell suspension (0.5 μl) was stained with 5 μl Annexin V-FITC and 10 μl propidium iodide (PI) (Beyotime, Biotechnology, Shanghai, China) under conditions void of light at room temperature for 15 min. Flow cytometry (BD Bioscience, U.S.A.) were subsequently applied to determine cell apoptosis at 488 nm. The experiment was repeated three times.

Statistical analysis
Statistical analysis was performed using the SPSS 21.0 software (IBM Corp. Armonk, NY, U.S.A.). The measurement data were expressed as mean + − standard deviation, with equality of variances and normal distribution tested. If the data subjected to normal distribution and equality of variances, comparisons among multiple groups were analyzed by one-way analysis of variance (ANOVA) or repeated-measures ANOVA, and the post hoc test was used for the pairwise comparison within a group. For the data with skew distribution or unequal variances, the Wilcoxon rank sum test was performed. A P-value < 0.05 was considered to be significantly different.

Successful establishment of PR-M(+) cells
The first step in our experiment was to establish the primary PR-M(+) uterine leiomyoma cells isolated from patients with uterine leiomyoma. Next, cellular morphology was observed and identified under a high-power microscope following immunohistochemistry. As depicted in Figure 1A, the cells were identified as uterine leiomyoma cells. The protein levels of PR-M, PR-A and PR-B were detected using Western blot analysis ( Figure 1B), which revealed that in the PR-M(+) cells, PR-M was highly expressed and PR-A/B was not expressed. The normal endometrial stromal cells served as the control, with our investigations revealing that the PRA/B antibody was effective [22]. The results obtained were indicative of the successful establishment of primary uterine leiomyoma cells and PR-M(+) cells.  (Figure  2A,B). Next, CCK-8 assay, MDC staining assay, Western blot analysis and flow cytometry were adopted to detect the cell viability, cell autophagy, expression of autophagy-related proteins (p62, LC3-I, and LC3-II) and cell apoptosis, respectively as illustrated in Figure 2C Figure 2F,G). The results obtained suggested that PR-M could exert an inhibitory role in the autophagy and apoptosis of primary uterine leiomyoma cells, which might be promoted by PR-M down-regulation.

PR-M inhibits the PR-M(+) cell autophagy and apoptosis via regulation of Bcl-2/Beclin1 axis
As the inhibitory role of PR-M in cell autophagy and apoptosis of PR-M(+) cells was demonstrated in the former experiments, we subsequently clarified whether the Bcl-2/Beclin1 axis is involved this process. Initially, Western blot analysis was employed to determine the protein level of Bcl-2 and Beclin1 ( Figure 3A). The results revealed that PR-M depletion decreased protein level of Bcl-2, and elevated the protein level of Beclin1 (P<0.05). In contrast, protein level of Bcl-2 was elevated in PR-M(+) cells with PR-M up-regulation, along with reduced protein level of Beclin1 (P<0.05). Second, a Co-immunoprecipitation (Co-IP) assay was performed to verify the interaction between Bcl-2 and Beclin1 in PR-M(+) cells. As depicted in Figure 3B, in the PR-M(+) cells infected with pSIH1-H1-copGFP expressing shRNA for PR-M, the interaction between Bcl-2 and Beclin1 was weakened, which was enhanced in those infected with pLV-EGFP expressing PR-M. Collectively, these results suggested that the Bcl-2/Beclin1 axis might involve in the inhibitory effect of PR-M on autophagy and apoptosis of primary uterine leiomyoma cells.

PRA promotes the PR-M(+) cell autophagy and apoptosis
With the results in the above section detailing the inhibitory role of PR-M in the PR-M(+) cell autophagy and apoptosis, we examined the hypothesis that PRA may have a reverse effect. In order to verify this, we treated the PR-M(+) cells using MIF at different concentrations, which is a type of PRA. Next, cell viability was evaluated using a CCK-8 assay ( Figure 4A). Our results revealed that cell viability was significantly decreased in PR-M(+) cells with MIF treatment, which was decreased most obviously in those with H-MIF. Next, MDC staining revealed that MIF enhanced the punctate green fluorescence intensity around nucleus, which exhibited the most significant increase following H-MIF treatment ( Figure 4B). Western blot analysis was subsequently employed to further determine the protein level of autophagy-related proteins (p62, LC3-I, and LC3-II). The results obtained ( Figure 4C Figure 4D,E). Above findings highlighted a facilitation effect of PRA in autophagy and apoptosis of primary uterine leiomyoma cells, which might be enhanced with increasing of the concentration.

PRA promotes the PR-M(+) cell autophagy and apoptosis via the Bcl-2/Beclin1 axis
After determining the role of PRA (MIF) in the autophagy and apoptosis of PR-M(+) cells, we then performed Western blot analysis and Co-IP in order to investigate whether Bcl-2/Beclin1 axis involved in this process. At first, Western blot analysis demonstrated that MIF treatment inhibited protein level of Bcl-2 and promoted protein level of Beclin1, which were changed most obviously in the PR-M(+) cells with H-MIF treatment (P<0.05, Figure 5A). Next, the interaction between Bcl-2 and Beclin1 in PR-M(+) cells treated with MIF was verified by Co-IP assay, which revealed that the interaction between Bcl-2 and Beclin1 was weakened in the PR-M(+) cells with L-MIF, M-MIF, and H-MIF, and weakened most significantly in those with H-MIF ( Figure 5B). The results obtained highlighted the potential involvement of the Bcl-2/Beclin1 axis in the promotion of PRA regarding the autophagy and apoptosis of primary uterine leiomyoma cells.

The PRA-modulated Bcl-2/Beclin1 axis involves in the PR-M(+) cell autophagy and apoptosis
In order to further clarify whether the PRA-modulated Bcl-2/Beclin1 axis is involved in the autophagy and apoptosis of PR-M(+) cells, the PR-M(+) cells were treated with combination of pLV-EGFP expressing Bcl-2 and H-MIF. RT-qPCR and Western blot analysis were employed to detect the transfection efficiency of Bcl-2, the results of which revealed that the mRNA and protein expression of Bcl-2 was significantly increased after oe-Bcl-2 treatment (P<0.05) ( Figure 6A,B), highlighting the successful establishment of pLV-EGFP expressing Bcl-2, which were used for subsequent experimentation. The MDC staining results revealed there to be a reduction in the punctate green fluorescence intensity around nucleus in the PR-M(+) cells following Bcl-2 overexpression and H-MIF treatment in comparison to H-MIF treatment alone ( Figure 6C). Moreover, Western blot analysis ( Figure 6D) was performed to determine the protein level of autophagy-related proteins, while flow cytometry combined with Western blot analysis ( Figure  6E,F) was used to determine the rate of cell apoptosis as well as the expression of activated Caspase-3. Our results revealed that compared with H-MIF treatment alone, co-treatment of overexpressed Bcl-2 and H-MIF increased the protein level of p62 and decreased levels of LC3-II/(LC3-I + LC3-II), coupled with a reduced cell apoptosis rate and activated Caspase-3 (P<0.05). Next, the protein level of Bcl-2 and Beclin1 and the interaction between Bcl-2 and Be-clin1 were evaluated by Western blot analysis and Co-IP, respectively. The results obtained displayed that Bcl-2 was notably elevated, while that of Beclin1 was reduced in PR-M(+) cells after co-treatment of overexpressed Bcl-2 and H-MIF (P<0.05, Figure 6G). As displayed in Figure 6H, the interaction between Bcl-2 and Beclin1 was enhanced after the co-treatment of overexpressed Bcl-2 and H-MIF. Collectively, the above results provided further evidence that the PRA-modulated Bcl-2/Beclin1 axis could be involved in promoting the autophagy and apoptosis of primary uterine leiomyoma cells.

Discussion
PRs are the most vital receptors that bind reproductive hormone, progesterone. They collaborate to help regulate menstruation, sexual development and reproduction in females [23]. PRs have various isoforms and have been shown to be capable of reversing hormone stimulation functions [11,12]. In the current study, our results revealed that MIF, as one antagonist of progesterone receptor, is capable of inhibiting the autophagy and apoptosis of PR-M(+) uterine leiomyoma cells by regulating Bcl/Beclin1 axis. The findings not only present a novel molecular function of PR-M in hysteromyoma, but also a potential therapeutic role that MIF could play against uterine leiomyoma. The first critical finding in our research was that it was PR-M not PR-A/B expressed specifically in primary uterine leiomyoma cells. The next important finding was that PR-M inhibited the autophagy and apoptosis of uterine leiomyoma cells through the Bcl-2/Beclin1 axis. Bcl-2 is a regulator protein that control cellular apoptosis [24]. It has been demonstrated that damage to the Bcl-2 gene can result in a variety of cancers, including breast cancer, prostate cancer, lung cancer, melanoma and chronic lymphocytic leukemia. For example, the co-overexpression of Bcl-2 and Myc can result in aggressive B cell malignancies [25]. The overexpression of Bcl-2 has been shown to lead to drug-resistance of Borrelidin in breast cancer cells, underlining the prognostic value of Bcl-2 in cancer [26]. Beclin1 is an important protein implicated in autophagy of cells, which usually interacts with the Bcl-2 protein. Beclin1 is also a tumorigenic regulator protein. Ovarian cancer cells with up-regulated Beclin1 show increased level of cellular autophagy and are much more sensitive to chemotherapy than normal ones. In lung cancer cells, Beclin1 and LC3 have been reported to exhibit down-regulated levels with the result of decreased autophagy level of cancer cells [27]. Additionally, norcantharidin was demonstrated to up-regulate the expression of Beclin1 by suppressing miR-129-5p, which then leads to an increase in the autophagy of prostate cancer cells [28]. Collectively, PR-M can regulate programmed-death of cancer cell via Bcl-2/Beclin1 axis.
Finally, our results demonstrated that the antagonist of progesterone receptor, MIF, can boost programmed-death of PR-M-positive uterine leiomyoma through the Bcl-2/Beclin1 axis. Although PR-M is a truncated form of a progesterone receptor, and it still contains a hinge and hormone-binding domain, which grants progesterone-binding capability for itself [10]. Currently, MIF, which was initially employed as an abortion drug in early pregnancy, has been increasingly investigated as a potential cancer treatment option. MIF has been shown to repress the proliferation of cancer stem cells in triple-negative breast cancer via down-regulation of KLF5 [29]. Furthermore, MIF has been shown to combine with proteasome or lysosome inhibitors in addition to killing ovarian cancer cells by elevating autophagy-related proteins. Given the possible benefit in cancer treatment, a previous study presented a comprehensive report highlighting the safety and effectiveness of the simultaneous usage of aspirin, lysine, MIF, and doxycycline against cancer metastasis [30]. Hence, although MIF was found to block cancer cell cycle by binding with PR-M, a more accurate elucidation of the molecular regulation between PR-M and Bcl-2 has yet to be acquired. Further studies are required to elucidate the mechanism associated with MIF medication.
In conclusion, the key findings of the current study highlight the therapeutic value of MIF in the treatment of PR-M-positive uterine leiomyoma, which results in the autophagy and apoptosis of cancer cells by regulating Bcl-2/Beclin1 axis ( Figure 7). However, considering that long term use of MIF may lead to inflammation of uterine or even un-pregnancy of women, it is necessary to conduct more studies to raise safety of MIF medication.