- Email: [email protected]
AKT pathway
Journal Pre-proof miR-92a promotes progesterone resistance in endometriosis through PTEN/AKT pathway
Manchao Li, Jintao Peng, Yanan Shi, Peng Sun PII:
S0024-3205(19)31118-X
DOI:
https://doi.org/10.1016/j.lfs.2019.117190
Reference:
LFS 117190
To appear in:
Life Sciences
Received date:
12 September 2019
Revised date:
4 November 2019
Accepted date:
14 December 2019
Please cite this article as: M. Li, J. Peng, Y. Shi, et al., miR-92a promotes progesterone resistance in endometriosis through PTEN/AKT pathway, Life Sciences(2019), https://doi.org/10.1016/j.lfs.2019.117190
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© 2019 Published by Elsevier.
Journal Pre-proof 1 miR-92a Promotes Progesterone Resistance in Endometriosis Through PTEN/AKT Pathway Manchao Li§*, Jintao Peng§, Yanan Shi, Peng Sun The Department of Reproductive Medicine Center, The Sixth Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, China Both authors contributed equally to this work and should be considered as equal first
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coauthors.
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*Corresponding author
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Manchao Li
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The Department of Reproductive Medicine Center, The Sixth Affiliated Hospital of Sun Yat-Sen University, No. 26 Erheng Road, Yuancun Street, Tianhe District, Guangzhou
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510000, Guangdong, China
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Tel: +86-18312025840
Email: [email protected]
Authors email Manchao Li
email: [email protected]
Jintao Peng
email: [email protected]
Journal Pre-proof 2 email: [email protected]
Peng Sun
email: [email protected]
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Yanan Shi
Journal Pre-proof 3 Abstract The alteration of PTEN expression may be a vital part of the pathological and physiological mechanisms in infertility-related with endometriosis. However, the potential mechanisms underlying abnormal expression of PTEN and its role in progesterone-resistant endometriosis have not been thoroughly elucidated. In this study,
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our data showed the PTEN messenger RNA (mRNA) level and protein expression was reduced in progesterone-resistant endometriosis tissue and primary stomal cells. Low
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levels of PTEN in endometrial stromal cells led to higher cell proliferation and resistance
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to progesterone. In terms of PTEN suppression in progesterone-resistant endometriosis,
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the mRNA level of miR-92a was correlated negatively with PTEN level. Transfection of
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miR-92a mimic reduced PTEN expression and made the stromal cells more resistant to progesterone treatment. Inhibition of miR-92a by its antagomir had the opposite effects.
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Results of the luciferase reporter assay for the 3′-nontranslated region suggested that
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miR-92a directly modulated PTEN levels. Moreover, miR-92a inhibition by its antagomir enhanced the therapeutic effect of progesterone, which suppressed stromal cell
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proliferation, and reduced the formation of ectopic lesions in the mouse model of endometriosis. Hence, this study revealed that miR-92a contributed to the development of progesterone resistant endometriosis by suppression of PTEN expression, and modulation of miR-92a might be a potential medical method of treating endometriosis. Keywords: miR-92a; mRNA; PTEN; Endometriosis
Journal Pre-proof 4 Introduction Endometriosis is considered as a gynecological disorder, and defined as the growth of stroma and glands of the endometrium in extrauterine locations, mainly the rectovaginal septum, ovaries, and pelvic peritoneum (Bulun, 2009, Giudice, 2010). Endometriosis affects 6% to 10% of women in child-bearing age, and the incidence is 35% to 50% among those who suffer from chronic pelvic pain and infertility, which has a negative
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effect on the quality of life and on health (Moradi et al. , 2014). Current therapies include
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surgery, medicine, and combined treatment (Giudice, 2010). After surgical removal of
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endometriosis, 30% to 50% of patients experience disease recurrence in 3 to 5 years. The
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most surprising phenomenon is the reappearance of endometriosis in women with both
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uterus and ovaries excised (Bulun, 2009, Guo and Olive, 2007). In women of childbearing age, the therapy with gonadotropin-releasing hormone analogs, androgenic
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agents, progesterone, or oral contraceptives can be used only in the short term to maintain
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low estrogen levels, because of adverse effects, such as bone density loss and pseudomenopause (Bulun, 2009, Giudice, 2010, Guo and Olive, 2007). After low-
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estrogen therapy is discontinued, the rate of recurrence within a year is as high as 60% (Guo and Olive, 2007). Therefore, current therapies cannot meet the goal of reducing the reappearance of endometriosis and have an adverse influence on women’s reproductive health and pregnant ability. We thus investigated some underlying signal pathways related to nonsteroidal or nonestrogen targets for treating endometriosis. Recent studies of linkage and genomewide associations have revealed a strong connection between endometriosis and the 10q23-26, 7p15.2, and 9p21 loci. PTEN (phosphate and tension homolog deleted on chromosome 10) is a tumor-suppressing gene
Journal Pre-proof 5 with a double-specificity phosphatase at the 10q23 locus. Via a certain AKT-dependent pathway, this gene could repress cellular division and promote apoptosis (Sansal and Sellers, 2004). Immunohistochemistry assays demonstrated that the decrease in PTEN levels in normal endometrial glands (“PTEN-null glands”) appeared in 43% of samples from healthy women experiencing premature menopause. Follow-up tests after approximately 1 year demonstrated that PTEN-null glands could still be identified in 83%
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of the women (Mutter et al. , 2001). With mutation, deletion, or inactivation of PTEN, the
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expression of the PTEN protein is downregulated, and its activity is decreased. Therefore,
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the ability of PTEN protein to inhibit tumor cell proliferation, adhesion, migration, and
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angiogenesis is weakened, which thus promotes the development of tumors (Stahl et al. , 2003). Endometriosis, a largely benign, chronic inflammatory disease, is an independent
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risk factor for endometrioid and clear cell epithelial ovarian tumors. A previous study
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(Forster et al. , 2011) showed that the occurrence and development of endometrial diseases are closely related to the functional inactivation of PTEN. In endometriosis, it
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was demonstrated that the AKT pathway, in the downstream of PTEN, is overactive in
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comparison with endometrial cells from endometriosis-free patients (Yin et al. , 2012). However, the mechanism of PTEN protein inactivation in progestin treatment for endometriosis remains unclear. We hypothesized that low expression of PTEN protein also leads to progesterone resistance in endometriosis and that the pathogenesis of endometriosis might involve other potential mechanisms that modulate PTEN levels. Micro-RNAs (miRNAs) are small RNAs located in the noncoding areas of DNA that could prevent translation of or even degrade the target genes by linking to the 3′-untranslated region (UTR) in mRNAs
Journal Pre-proof 6 (Calin et al. , 2004). The modulation of PTEN is thought to be dependent on miRNA (Johnson et al. , 2005). In this study, we demonstrated that the low expression of PTEN is one of the sources of progesterone resistance in human endometriosis. The miR-92a is responsible for the upstream regulation of PTEN by targeting its 3′-UTR and contributes to progesterone resistance. Suppression of miR-92a can overcome progesterone resistance both in cells and in the whole organism by suppressing the proliferation
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process in stromal cells.
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Materials and Methods
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Tissue samples
The Sixth Affiliated Hospital of Sun Yat-Sen University Institutional Review Board
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approved our research. All participating patients were fully informed about the study and
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provided written consent to participate. During salpingo-oophorectomy or ovarian
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cystectomy, endometrial cyst walls were obtained from patients suffering from endometriosis in the Sixth Affiliated Hospital of Sun Yat-Sen University. All the patients had undergone progesterone therapy and were divided into two groups: those experienced pelvic pain relief after progesterone treatment (Responsive, n = 11), and those who did not have pelvic pain relief after progesterone treatment (Resistant, n = 12). Detailed information about the patients is listed in Table 1. The histological results of the samples were provided by pathologists.
Journal Pre-proof 7 Table
1.
The
information
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patients
endometriosis
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Stromal cell isolation and cell culture
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The stromal cells of the patients were isolated as described previously (Ryan et al. , 1994).
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In cell culture, we used Dulbecco’s modified Eagle’s medium (DMEM)/nutrient mixture
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F-12 in a ratio of 1:1 (Invitrogen, Carlsbad, Calif.) supplemented with fetal bovine serum
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(10%) and streptomycin, 100 U/mL, with penicillin, 100 U/mL, in an environment of 37°C with a wet atmosphere and CO2 (5%). Laboratory-grown human endometrial
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stromal cells (SHT290; Kerafast, Boston) were cultured in DMEM combined with
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epidermal growth factor (20 ng/mL; Becton Dickinson, Franklin Lakes, N.J.), glutamine
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(2 mM), HyClone fetal bovine serum (2%; Thermo Fisher Scientific, Waltham, Mass), and streptomycin, 100 U/mL, with penicillin, 100 U/mL, in a wet environment of 37°C supplemented with CO2 (5%). For both patients’ cells and the SHT290 cells, we changed the medium every 3 days. After the seventh pass, the cells were prepared for the tests described in the next sections. Specific concentrations of progesterone (Sigma-Aldrich, Shanghai) were used to treat cells. Cell viability and proliferation
Journal Pre-proof 8 Primary stromal cells or SHT290 cells were seeded in 96-well plates at a concentration of 5 × 104 cells in each well, and the cells were left overnight for attaching. Afterward, the cells were rinsed with phosphate buffer solution and then incubated in DMEM/F-12 (1:1; phenol red-free) supplemented with fetal bovine serum (10%) and streptomycin, 100 U/mL, with penicillin, 100 U/mL, and also progesterone in different concentrations for 24 hours. To detect cellular viability, we used the MTT assay kit (Sigma-Aldrich) in
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accordance with the manufacturer’s protocols, and we measured fold change in relation
incorporation
immunoassay
(Sigma-Aldrich)
in
accordance
with
the
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(BrdU)
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to vehicle control. To detect cell proliferation, we used the 5-bromo-2′-deoxyuridine
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manufacturer’s protocols, and we measured fold change in relation to vehicle control.
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Cell transfection and grouping
All the sequences we used were obtained from Shanghai Sangon Biological Engineering
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Technology & Services Co., Ltd. (Shanghai) as described in an earlier report (Li et al. ,
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2018). Before cell transfection, we cultured the cells in 6-well plates at a concentration of
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2 × 106 cells in each well. The confluence of the cells was 70% to 80% when the transfection was conducted. In accordance with the manufacturer’s protocols for Lipofectamine 2000 kits (Invitrogen), Gibco Opti-MEM medium without serum (250 μL; Thermo Fisher Scientific) was combined in four separate mixtures—with 100 pmol of miR-92a mimic, with PTEN small interfering RNAs (siRNAs)-, with miR-92a antagomir, and with normal control—with ultimate concentrations of 50 nmol/L. Afterward, the mixtures were cultured at normal room temperature for 5 minutes. Then, Gibco OptiMEM medium without serum (250 μL) was mixed with Lipofectamine 2000 (5 μL) and cultured at normal room temperature for 5 minutes. The two mixtures mentioned
Journal Pre-proof 9 previously were combined, cultured for 20 minutes at normal room temperature, and then added to the cells. After 6 hours of transfection, we replaced the mixtures with the complete medium and cultured the cells in a 37°C incubator with a wet atmosphere supplemented with CO2 (5% ) and room air for 24 to 48 hours in order to conduct the next experiments.
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Reverse transcription-quantitative polymerase chain reaction
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In accordance with the protocol for using TRIzol reagent (Invitrogen), we extracted total
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RNA from endometriosis tissues and cells so as to measure the purity and concentration of the RNA. The RNA we extracted was kept in a
80°C refrigerator for the next assays.
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In accordance with the protocols for the complementary DNA (cDNA) synthesis kit
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(TaqMan MicroRNA Reverse Transcription Kit; Thermo Fisher Scientific), we added the
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sterile water to dilute the cDNA we obtained from reverse transcription (diluting ratio, 1:5), and then preserved them in a 20°C refrigerator for next assays. The primers for
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PTEN and miR-92a were provided by Shanghai Biowing Applied Biotechnology Co.,
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Ltd. (Shanghai). The polymerase chain reaction (PCR) system (20 μL in total) included 0.4 μL of 50× Rox Reference Dye, 2 μL of cDNA (diluted 1:5), 2 μL of Universal Adaptor PCR Primer (2 μmol/L), 2 μL of All-in-One miRNA Q-PCR Primer (2 μmol/L), 20 μL of DNase/RNase-free H2O, and 10 μL of 2 × All-in-One Q-PCR Mix. The amplification conditions consisted of predenaturation at 95°C for 10 minutes; denaturation at 95°C for 40 cycles, each lasting 10 seconds; annealing at 60°C for 20 seconds; and the elongation process at 72°C for 34 seconds. We used the Applied Biosystems 7500 quantitative PCR system (Applied Biosystems, New York) to conduct reverse transcription-quantitative -PCR. The β-actin acted as an internal reference in the
Journal 10 Pre-proof detection of miR-92a in the measurement of PTEN. We used the 2−ΔΔCt method to measure the relative expression of the target genes. The primers sequences were as described in an earlier report (Li et al., 2018). Immunoblotting The patients’ stromal cells and the SHT290 cells were rinsed with ice-cold phosphate
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buffer solution, and we used M-PER Mammalian Protein Extraction Reagent (Thermo
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Fisher Scientific, Shanghai) with phosphatase inhibitors and protease (both from Sigma-
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Aldrich, St. Louis) on ice to obtain cell lysates. The Micro BCA Protein Assay Kit (Thermo Fisher Scientific) was used to determine the total protein concentrations.
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Proteins in equal amounts were separated on 10% polyacrylamide gel and then
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transferred onto polyvinyl difluoride membranes. Then, 5% bovine serum albumin in trisbuffered saline with 0.1% Tween 20 (TBST; Sigma-Aldrich) was used to block the
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membranes for 1 hour at room temperature. Afterward, the membranes were incubated
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for 1 night in a 4°C refrigerator with primary antibodies (in 1% bovine serum albumin)
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against β-actin (Sigma-Aldrich), Ki-67 (Cell Signaling Technology, Danvers, Mass), PTEN, total AKT, and phospho-AKT (Ser473). The next day, the membranes were washed with TBST and then incubated with anti-mouse or goat anti-rabbit secondary antibodies bound with horseradish peroxidase (concentration, 1:4000) for 1 hour. Then, with a kit for chemiluminescent detection (Thermo Fisher Scientific), a Fujifilm LAS3000 Imager (Fujifilm, Tokyo) was used to take digital pictures of the Western blots. Immunohistochemistry assay
Journal 11 Pre-proof Paraformaldehyde (4%) was used for tissue fixation, and then the tissues were embedded in paraffin. We cut the embedded tissues into 4-μm-thick sections and placed them onto the glass slides. The sections were then deparaffinized and rehydrated, and we performed antigen restoration by heating the sections in citrate buffer (pH, 6.0). By culturing the sections in 0.03% H2O2 for 10 minutes, we blocked the activity of endogenous peroxidase. Dako protein block (Agilent Technologies, Santa Clara, Calif.) was applied
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to the sections for 30 minutes, and then the slides were rinsed with TBST and cultured
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with the primary antibodies against Ki-67 (1:100; Cell Signaling Technology) overnight
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in a 4°C wet box. The next day, the slides were washed with TBST, and then we applied
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secondary antibodies conjugated with horseradish peroxidase for 30 minutes. Diaminobenzidine tetrahydrochloride was used as a substrate to detect horseradish
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Luciferase reporter assay
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peroxidase activity, and the slides were counterstained with hematoxylin.
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The dual-luciferase reporter experiment was conducted for further gene identification.
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We cloned and amplified the full length of the 3′-UTR of PTEN. We subcloned the PCR products and inserted them in the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega Corporation, Madison, Wisc.), and we named them “PTEN-wt” (PTENwide type). Afterward, we conducted mutagenesis directed by site in the combined site in miR-92a and in the target gene obtained from bioinformatics software so as to establish a carrier for the PTEN mutant (“PTEN-mut”). We used the pRL-TK carrier that expressed renilla luciferase (Takara Holdings Inc., Kyoto) as the internal control in order to eliminate the differences in efficiency and cell amounts of transfection. The miR-92a antagomir and mimics were separately transfected into SHT290 cells, together with
Journal 12 Pre-proof carriers of luciferase reporter, and the activity of the dual-luciferase reporter gene has been measured with the methods provided by Promega Corporation. Induction of endometriosis in mice Animals were kept in a specific room in which care was based on the instructions from The Sixth Affiliated Hospital of Sun Yat-Sen University's institution of the use and care
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for experimental animals. By placing endometrial tissue in the animals’ peritoneal cavity,
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we established the endometriotic lesions in vivo. Tribromoethanol (Avertin) injection
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was used to produce deep anesthetization in the animals. We made a 1-cm midventral incision for laparotomic access to the peritoneal cavity, identified the intestine and uterus,
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excised the left uterine horn, and placed it in Basal Medium Eagle (Invitrogen) in a petri
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dish. The mean weight of the samples was 56.70 ± 5.47 mg. We cut the uterine horn open along the long axis and cut it into small pieces. The pieces were resuspended in Basal
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Medium Eagle (0.5 mL) and placed back into the abdominal cavity in the same mouse
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whose uterus has been excised to perform the autologous implantation. Then we gently
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massaged the abdomen to make the tissues fully disperse. 17-Estradiol pellets (SE-121; Innovative Research of America, Sarasota, Fla.), 0.36 mg/day, were implanted subcutaneously into the mice for 60 days to promote the formation of lesions similar to those of endometriosis. Mice were treated with progesterone, 50 mg/kg, with or without anti-miR92a, 10 μM/kg subcutaneously, once every 4 days starting on the fourth day before lesion formation was induced. After 4 weeks, the experimental mice were sacrificed. We opened the abdominal cavity and then counted and excised the endometriosis-like lesions. Those lesions and the uterine tissues were fixed in paraformaldehyde (4% in phosphate buffer solution) for further histological analysis.
Journal 13 Pre-proof Statistics analysis SPSS 18.0 software (SPSS Inc., Chicago) was used for statistical analysis. Chi-square tests were used to compare data between groups. The measurements were recorded as means ± standard deviations. If the data displayed homogeneous variance and were regularly distributed, we conducted t-tests to compare the two groups. P values lower
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than 0.05 were considered statistically significant.
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Results
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PTEN level was suppressed in women with progesterone-resistant endometriosis
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To detect the expression of PTEN and discover whether resistance to progesterone was present in women with endometriosis, we conducted quantitative reverse transcription
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PCR to measure levels of PTEN mRNA, using the endometrial samples from 11 women
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with progesterone-responsive endometriosis and 12 women with progesterone-resistant
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endometriosis. The levels of PTEN mRNA were remarkably lower in progesteroneresistant endometriosis samples than in the samples of patients with endometriosis, which can be relieved by progesterone treatment (Figure 1A). Consistently, the results of the Western blot also demonstrated that the expression of PTEN was lower in the patients with progesterone-resistant endometriosis (Figure 1B). After isolating the primary stromal cells from the tissue in progesterone-responsive and progesterone-resistant patients, we found that the progesterone-resistant cells had higher proliferation ability than did the progesterone-responsive cells (Figure 1C). Progesterone treatment suppressed the proliferation of progesterone-responsive stromal cells but showed no
Journal 14 Pre-proof effect on the proliferation process of progesterone-resistant stromal cells (Figure 1C). In further confirmation of the results, the BrdU assay and Ki-67 Western blot produced similar results (Figure 1D, E). In the progesterone-resistant stromal cells, the expression of PTEN was suppressed, but AKT was hyperactivated (Figure 1E). Moreover, the induction of PTEN and suppression of AKT by progesterone in responsive stromal cells were also abolished in the resistant stromal cells (Figure 1E). Therefore, our data
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indicated that PTEN expression was lower in the women with progesterone-resistant
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endometriosis.
Figure 1. PTEN expression was lower in progesterone resistant endometriosis. (A) The messenger RNA level of PTEN in endometrial biopsy specimens from patients with progesterone-responsive endometriosis (n = 11) and those with progesterone-resistant endometriosis (n = 12). (B) Representative Western blot of PTEN in endometrial biopsy specimens from patients with progesterone-responsive and patients with progesterone-
Journal 15 Pre-proof resistant endometriosis. The primary cells derived from these patients were treated with progesterone, 1 μM, for 48 hours. The cell proliferation was analyzed with MTT tests (C) and 5-bromo-2′-deoxyuridine (BrdU) assay (D). (E) The level of p-AKT, PTEN, and Ki67 has been analyzed with Western blot. Each experiment was repeated 3 times. N,
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p>0.05; *, p<0.05; **, p<0.01.
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Abnormal expression of PTEN mediated the progesterone resistance in endometriosis
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To evaluate the function of PTEN in progesterone resistance, a human endometrial
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stromal cell line, SHT290, was used in the following studies. We found that the transfection of PTEN siRNA in SHT290 cells suppressed the expression of PTEN
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(Figure 2A) but increased the expression of Ki-67, as well as activation of AKT (Figure
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2A), which suggested the higher proliferation of stromal cells after PTEN knockdown. The MTT assay and BrdU analysis also indicated that the absence of PTEN promoted the
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proliferation of SHT290 cells (Figure 2B, C). The depletion of PTEN also ameliorated
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the ability of progesterone to inhibit the proliferation of SHT290 cells (Figure 2B, C). To further confirm the effect of PTEN, we treated the progesterone resistant primary stromal cells with AKT inhibitor, LY294002 because activation of AKT occurs downstream of PTEN loss. As predicted, LY294002 suppressed the proliferation ability of progesteroneresistant stromal cells and also increased progesterone resistance in stromal cells (Figure 2D, E). These findings suggested that dysregulation of PTEN contributed to progesterone resistance in endometriosis.
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Figure 2. Absence of PTEN contributed to progesterone resistant in endometriosis. (A) The levels of PTEN and Ki-67 in SHT290 cells that were transfected with PTEN siRNA.
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The SHT290 cells transfected with PTEN siRNA were treated with 1 μM progesterone.
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The proliferation of cells was analyzed with MTT assay (B) and 5-bromo-2′deoxyuridine (BrdU) assay (C). The primary stromal cells from patients with
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progesterone-resistant endometriosis were treated with progesterone, 1 μM, with or
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without the combination of LY294002, 1 μM. The proliferation of cells was analyzed with MTT tests (B) and BrdU assay (C). Each experiment was repeated 3 times. N, p>0.05; *, p<0.05; **, p<0.01.
Journal 17 Pre-proof An increased miR-92a level is related to downregulated PTEN expression in progesterone-resistant endometriosis MiRNA was reported to modulate PTEN expression (Li et al., 2018). To reveal the potential mechanism of low PTEN expression in progesterone-resistant endometriosis, we analyzed the expression of several miRNAs that were reported to target PTEN,
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including miR-92a (Li et al., 2018), miR-26a (Xiong et al. , 2019), miR-130b, miR-494 (Lu et al. , 2019), miR-103 (Jiang et al. , 2019), and miR-21 (Liu et al. , 2019). The
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results from quantitative reverse transcription PCR experiments indicated that the miR-
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92a level increased remarkably in the patients with progesterone-resistant endometriosis
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(Figure 3A). In contrast, no great difference has been shown by other miRNAs between
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progesterone-responsive and progesterone-resistant endometriosis. The expression of miR92a was also negatively related to the mRNA level of PTEN in the 23 patients in this
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study (Figure 3B). On the basis of our results, we performed further studies on cells to
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explore the potential mechanism in the opposite relation between miR-92a and PTEN levels. Similarly, we discovered that the level of miR-92a was also higher in the primary
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stromal cells derived from patients with progesterone-resistant endometriosis (Figure 3C). Therefore, our data suggested that miR-92a might be the upstream regulator of PTEN in progesterone-resistant endometriosis.
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Figure 3. The level of miR-92a has been increased in progesterone resistant
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endometriosis. (A) The messenger RNA level of micro-RNAs (miRNAs) in endometrial biopsy specimens from patients with progesterone-response endometriosis (n = 11) and patients with progesterone-resistant endometriosis (n = 12). (B) The correlation of miR92a level with the messenger RNA level of PTEN in patients with endometriosis (n = 23). (C) The miR-92a level in primary stromal cells from progesterone-responsive or progesterone-resistant patients. Each experiment was repeated 3 times. N, p>0.05; **, p<0.01.
Journal 19 Pre-proof miR-92a reduces the PTEN level in cells via aiming at the 3′-untranslated region To further verify that PTEN is the target for miR-92a in the downstream, we detected the PTEN mRNA level under miR-92a activation and inhibition. The transfection of stromal cells with miR-92a mimic dramatically increased the miR-92a level (Figure 4A). Conversely, transfection with miR-92a antagomir dramatically suppressed miR-92a expression (Figure 4A). Upon investigating the effect of miR-92a activation or inhibition
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on the mRNA level of PTEN, we found that overexpression of miR-92a suppressed
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PTEN expression, but inhibition of miR-92a could elevate the mRNA expression of
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PTEN (Figure 4B). To confirm PTEN as a target gene for miR-92a, we respectively
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inserted PTEN-mut and PTEN-wt sequences in the reporter plasmids. The miR-92a antagomir or mimics and PTEN-wt or PTEN-mut recombined plasmids were transfected
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together into the SHT290 cells with the luciferase reporter gene. Our results
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demonstrated that the miR-92a mimic suppressed the PTEN-wt luciferase activity, which was enhanced by miR-92a antagomir co-transfection (Figure 4C). However, the co-
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transfection of the miR-92a mimic or antagomir showed no remarkable influences on the
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activity of luciferase on PTEN-mut plasmids (Figure 4D). The miR-92a mimic also suppressed the protein level of PTEN, but its antagomir enhanced the expression of PTEN in SHT290 cells (Figure 4E). These findings indicate that PTEN could be one of the target genes for miR-92a.
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Figure 4. MiR-92a expression targeted the 3′-untranslated region (UTR) in PTEN. (A) The miR-92a level in SHT290 cells that were transfected by antagomir (right) or miR92a mimic (left). (B) The messenger RNA level of PTEN in SHT290 cells that were transfected by miR-92a antagomir or mimic. (C) The activity of PTEN-wide-type luciferase reporter gene in SHT290 cells that were transfected by miR-92a antagomir or mimic. (D) The activity of 3′-UTR mutant PTEN luciferase reporter gene in SHT290
Journal 21 Pre-proof cells that were transfected by miR-92a antagomir or mimic. (E) PTEN and phospho-AKT level in SHT290 cells that were transfected by miR-92a antagomir or mimic. Each experiment was repeated 3 times. N, p>0.05; **, p<0.01; ***, p<0.001.
MiR-92a overexpression suppresses the effect of progesterone in vitro
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To identify the relationship between miR-92a and PTEN levels in progesterone resistance,
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we studied the effects of this relationship on SHT290 cell proliferation. Transfection with
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miR-92a promoted the proliferation of SHT290 cells and made them resistant to
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progesterone (Figure 5A). Results of the BrdU assay also indicated that miR-92a overexpression promoted SHT290 cell proliferation (Figure 5B) and compromised the
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inhibitory effect of progesterone (Figure 5B). Conversely, transfection with miR-92a
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antagomir inhibited the proliferation of SHT290 cells and also enhanced the suppressive effects of progesterone (Figure 5C). The BrdU assay yielded similar results (Figure 5D).
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All these results suggested that miR-92a expression contributed to progesterone
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resistance in endometriosis.
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Figure 5. MiR-92a expression mediated the progesterone resistance in stromal cells. The
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SHT290 cells that were transfected by miR-92a mimic were treated with progesterone, 1 μM. The cell proliferation ability was analyzed with MTT tests (A) and 5-bromo-2′deoxyuridine (BrdU) assay (B). The SHT290 cells transfected with miR-92a antagomir were treated with progesterone, 1 μM. The cell proliferation was analyzed by MTT assay (C) and BrdU assay (D). Each experiment was repeated 3 times. N, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001.
Journal 23 Pre-proof Inhibition of miR-92a restored endometriosis to progesterone treatment in vivo To clarify the effect of miR-92a inhibition on progesterone resistance in endometriosis, we induced endometriosis in syngeneic mice. In these model animals, small endometrial cell groups without myometrium were placed in the peritoneum cavities of syngeneic female mice to help the cells randomly attach and grow. We excised the ovaries in those mice and then subcutaneously administered progesterone with or without anti-
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miR92a antagomir to these mice once every 4 days starting, on the fourth day before the
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induction of lesions. We discovered that miR-92a antagomir remarkably suppressed the
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miR-92a level (Figure 6A), which was suggestive of the efficiency of antagomir in
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animal models. The number and volume of the abnormal lesions were calculated in the mice with the dissecting microscope, and then we conducted histological evaluations. All
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transplanted endometriotic lesions were found in the peritoneum. The progesterone or
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antagomir single treatment did not significantly reduce the number or volume of lesions (Figure 6B). However, the combined treatment significantly suppressed the formation
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and volume of the ectopic lesions (Figure 6B). Western blot revealed that miR-92a
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antagomir enhanced the PTEN level but suppressed AKT activation (Figure 6C). Furthermore, the Ki-67 immunochemistry staining also indicated that miR-92a antagomir enhanced the inhibitory effect of progesterone on the proliferation of cells (Figure 6D). Therefore, our data collectively indicated that the administration of miR-92a antagomir can restore the progesterone response in endometriosis in vivo.
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Figure 6. Inhibition of miR-92a enhanced the therapy effect of progesterone in vivo. (A)
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The messenger RNA level of miR-92a in ectopic tissue of mice with endometriosis after injection with miR-92a antagomir. (B) Mice were treated with the administration of miR-
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92a antagomir, 10 mM/kg, or progesterone, 50 mg/kg, or both when tissue injection was
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conducted and then once every 4 days for 4 weeks (n = 6 for each group). Lesions and the relative volume in the peritoneal cavity were measured. (C) The level of AKT, phosphoAKT, and PTEN in the ectopic tissues from different groups of mice. (D) The Ki-67 staining of ectopic lesions. Each experiment was repeated 3 times. N, p>0.05; **, p<0.01; ***, p<0.001.
Journal 25 Pre-proof Discussion The typical characters of endometriosis are infertility and severe and cyclic chronic pain in the pelvis. The causes of endometriosis-related infertility still have not been defined well, but the disease is caused by abnormal expression of genes in normal endometrial tissue and also correlated with progesterone resistance (Bulun, 2009, Fazleabas, 2010, Giudice, 2010). Progesterone therapy in the form of regular hormonal treatments helps
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alleviate symptoms and inhibits the reappearance of endometriosis (Mounsey et al. ,
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2006). However, treatment efficacy is only suitable for a specific group of patients.
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Multiple models have revealed that PTEN plays an important role in the action of
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progesterone on the endometrium, and loss of PTEN expression hinders embryonic
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implantation in the endometriosis patients (Guzeloglu-Kayisli et al. , 2003, Kim et al. , 2014). However, whether abnormal expression of PTEN in endometriosis contributes to
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progesterone resistance is still unknown. We found that decreased expression of PTEN in
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endometriosis led to progesterone resistance. We also identified a novel miRNA, miR92a, which was associated with PTEN suppression in endometriosis. The results from the
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cell and animal experiments suggested that inhibiting miR-92a increased the expression of PTEN and overcame progesterone resistance by suppressing the stromal cell proliferation. Thus MiR-92a could represent a new therapy for endometriosis. PTEN is a tumor suppressor gene that acts against the AKT/phosphatidylinositol 3-kinase (PI3K) signal pathway, inhibiting cell proliferation and survival rate (Risinger et al. , 1997, Steck et al. , 1997). Earlier research suggested that changes in the PTEN gene frequently occur in tissues from the endometrium of women who suffer from endometrial hyperplasia and endometriosis, as well as ovarian and endometrial tumors (Castiblanco et
Journal 26 Pre-proof al. , 2006, Dinulescu et al. , 2005, Mandai et al. , 2009). In ovaries from both humans and mice, the PI3K-PTEN-AKT-Foxo3 pathway was activated in samples from women with endometriosis. Administration of AS101 restored the proportion of primordial follicles in the ovaries of mice with endometriosis to control levels (Takeuchi et al. , 2019). In our study, we found that human patients with endometriosis, who had low PTEN expression, developed progesterone resistance. It was reported that progesterone treatment
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upregulated the expression of PTEN to suppress endometriosis proliferation (Mutter et
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al. , 2000). Our in vitro data also indicated that silence of PTEN in stromal cells enhanced
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cell proliferation and abolished the suppressive effect of progesterone (Fig. 2). Therefore,
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it is reasonable that low expression of PTEN results in progesterone resistance in
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endometriosis.
More importantly, accumulating evidence suggested that ovarian and endometrial tumors
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are usually identified in relation to endometriosis, which indicates that they represent
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malignant transformation (Dinulescu et al., 2005, McMenamin et al. , 1999). Thus women with endometriosis may have increased risks for multiple cancers of various
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kinds (Verri et al. , 2005). Hence, complete loss of or mutations in PTEN might result in the formation and growth of endometriosis, which might eventually turn into cancer. Our results about targeting miR-92a to reverse PTEN expression not only provided a new concept to overcome the progesterone resistance in endometriosis but also can protect patients with endometriosis from malignant transformation. Over a long time, many researchers have investigated the probable function of miRNA in endometriosis formation (Grechukhina et al. , 2012, Pei et al. , 2018). In this study, we noticed a remarkable elevation of the miR-92a level in progesterone-resistant
Journal 27 Pre-proof endometriosis, which was correlated with the suppression of PTEN expression. According to our data, the cells transfected with miR-92a mimics demonstrated decreased PTEN levels but enhanced AKT phosphorylation process. Some researchers have shown that miR-92a had a close association with human malignancy (Li et al., 2018). In gastric malignancy, miR-92a is considered oncogenic, with an important role in promoting cell proliferation by suppressing FBXW7, a tumor-suppressing gene (Liu et al. , 2016). In
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contrast, inhibiting miR-92a could dramatically impede cell growth and promote cell
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apoptosis, which has been studied in research (Sharifi et al. , 2014). A reduced PTEN
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level and an elevated miR-92a level were discovered in tissues from colorectal cancer,
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which implicated that they were negatively correlated (Ke et al. , 2015). Furthermore, miR-92a facilitates tumor development and inhibits immune activity by activating the
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MAPK/ERK signal pathway through the suppression of PTEN in cervical malignancy (Li
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et al., 2018). However, the expression of miR-92a in women who suffer from endometriosis is still unknown. In this study, we initially demonstrated that the
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proliferation of endometrial stromal cells was enhanced after the transfection of anti-
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PTEN siRNA or miR-92a mimic. Accordingly, our data revealed that the high expression of miR-92a suppresses the expression of PTEN, which results in progesterone resistance in endometriosis. These data revealed a novel function of miR-92a in endometriosis.
In conclusion, this research suggests that miR-92a is highly expressed in the progesterone-resistant endometriosis, which suppresses the expression of PTEN. Our findings reveal that the efficiency of progesterone in endometriosis therapy could be poor as a result of downregulated PTEN signal by miR-92a dysregulation, which suggests a potential application of miRNAs as biological therapy in the treatment of endometriosis.
Journal 28 Pre-proof Acknowledgements: None.
Conflicts of interest: The authors confirm that there are no conflicts of interest.
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Funding: This research did not receive any specific grant from funding agencies in the
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public, commercial, or not-for-profit sectors.
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Author contributions
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Study design/planning: Manchao Li, Jintao Peng
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Data collection/entry: Manchao Li, Jintao Peng
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Data analysis/statistics: Manchao Li, Jintao Peng, Yanan Shi, Peng Sun
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Data interpretation: Manchao Li, Jintao Peng, Yanan Shi, Peng Sun Preparation of manuscript: Manchao Li, Jintao Peng Literature analysis/search: Yanan Shi, Peng Sun
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[Pathogenic role of PTEN tumor suppressor gene in ovarian cancer associated to
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Dinulescu DM, Ince TA, Quade BJ, Shafer SA, Crowley D, Jacks T. Role of K-ras and
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Fazleabas AT. Progesterone resistance in a baboon model of endometriosis. Semin
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Forster MD, Dedes KJ, Sandhu S, Frentzas S, Kristeleit R, Ashworth A, et al. Treatment
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with olaparib in a patient with PTEN-deficient endometrioid endometrial cancer.
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Nat Rev Clin Oncol. 2011;8:302-6. Giudice LC. Clinical practice. Endometriosis. N Engl J Med. 2010;362:2389-98. Grechukhina O, Petracco R, Popkhadze S, Massasa E, Paranjape T, Chan E, et al. A polymorphism in a let-7 microRNA binding site of KRAS in women with endometriosis. EMBO Mol Med. 2012;4:206-17. Guo SW, Olive DL. Two unsuccessful clinical trials on endometriosis and a few lessons learned. Gynecol Obstet Invest. 2007;64:24-35.
Journal 30 Pre-proof Guzeloglu-Kayisli O, Kayisli UA, Al-Rejjal R, Zheng W, Luleci G, Arici A. Regulation of PTEN (phosphatase and tensin homolog deleted on chromosome 10) expression by estradiol and progesterone in human endometrium. J Clin Endocrinol Metab. 2003;88:5017-26. Jiang L, Qiao Y, Wang Z, Ma X, Wang H, Li J. Inhibition of microRNA-103 attenuates inflammation and endoplasmic reticulum stress in atherosclerosis through disrupting
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Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A, et al. RAS is
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Ke TW, Wei PL, Yeh KT, Chen WT, Cheng YW. MiR-92a Promotes Cell Metastasis of Colorectal Cancer Through PTEN-Mediated PI3K/AKT Pathway. Ann Surg Oncol.
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Kim TH, Yu Y, Luo L, Lydon JP, Jeong JW, Kim JJ. Activated AKT pathway promotes establishment of endometriosis. Endocrinology. 2014;155:1921-30.
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Li ZH, Li L, Kang LP, Wang Y. MicroRNA-92a promotes tumor growth and suppresses
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immune function through activation of MAPK/ERK signaling pathway by inhibiting PTEN in mice bearing U14 cervical cancer. Cancer Med. 2018. Liu C, Zhang Y, Chen H, Jiang L, Xiao D. Function analysis of rs9589207 polymorphism in miR-92a in gastric cancer. Tumour Biol. 2016;37:4439-44. Liu Z, Liang X, Li X, Liu X, Zhu M, Gu Y, et al. MiRNA-21 functions in ionizing radiation-induced epithelium-to-mesenchymal transition (EMT) by downregulating PTEN. Toxicol Res (Camb). 2019;8:328-40.
Journal 31 Pre-proof Lu Q, Liu T, Feng H, Yang R, Zhao X, Chen W, et al. Circular RNA circSLC8A1 acts as a sponge of miR-130b/miR-494 in suppressing bladder cancer progression via regulating PTEN. Mol Cancer. 2019;18:111. Mandai M, Yamaguchi K, Matsumura N, Baba T, Konishi I. Ovarian cancer in endometriosis: molecular biology, pathology, and clinical management. Int J Clin Oncol. 2009;14:383-91.
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McMenamin ME, Soung P, Perera S, Kaplan I, Loda M, Sellers WR. Loss of PTEN
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expression in paraffin-embedded primary prostate cancer correlates with high
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Moradi M, Parker M, Sneddon A, Lopez V, Ellwood D. Impact of endometriosis on women's lives: a qualitative study. BMC Womens Health. 2014;14:123.
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Mutter GL, Lin MC, Fitzgerald JT, Kum JB, Eng C. Changes in endometrial PTEN expression throughout the human menstrual cycle. J Clin Endocrinol Metab. 2000;85:2334-8. Pei T, Liu C, Liu T, Xiao L, Luo B, Tan J, et al. miR-194-3p Represses the Progesterone Receptor and Decidualization in Eutopic Endometrium From Women With Endometriosis. Endocrinology. 2018;159:2554-62.
Journal 32 Pre-proof Risinger JI, Hayes AK, Berchuck A, Barrett JC. PTEN/MMAC1 mutations in endometrial cancers. Cancer Res. 1997;57:4736-8. Ryan IP, Schriock ED, Taylor RN. Isolation, characterization, and comparison of human endometrial and endometriosis cells in vitro. J Clin Endocrinol Metab. 1994;78:6429. Sansal I, Sellers WR. The biology and clinical relevance of the PTEN tumor suppressor
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Sharifi M, Salehi R, Gheisari Y, Kazemi M. Inhibition of microRNA miR-92a induces
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through modulation of p63 expression. Mol Biol Rep. 2014;41:2799-808. Stahl JM, Cheung M, Sharma A, Trivedi NR, Shanmugam S, Robertson GP. Loss of
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Steck PA, Pershouse MA, Jasser SA, Yung WK, Lin H, Ligon AH, et al. Identification of
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mutated in multiple advanced cancers. Nat Genet. 1997;15:356-62. Takeuchi A, Koga K, Satake E, Makabe T, Taguchi A, Miyashita M, et al. Endometriosis triggers excessive activation of primordial follicles via PI3K-PTEN-Akt-Foxo3 pathway. J Clin Endocrinol Metab. 2019. Verri E, Guglielmini P, Puntoni M, Perdelli L, Papadia A, Lorenzi P, et al. HER2/neu oncoprotein overexpression in epithelial ovarian cancer: evaluation of its prevalence and prognostic significance. Clinical study. Oncology. 2005;68:154-61.
Journal 33 Pre-proof Xiong Y, Cao F, Hu L, Yan C, Chen L, Panayi AC, et al. miRNA-26a-5p Accelerates Healing via Downregulation of PTEN in Fracture Patients with Traumatic Brain Injury. Mol Ther Nucleic Acids. 2019;17:223-34. Yin X, Pavone ME, Lu Z, Wei J, Kim JJ. Increased activation of the PI3K/AKT pathway compromises decidualization of stromal cells from endometriosis. J Clin Endocrinol
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Metab. 2012;97:E35-43.
Manchao Li, Jintao Peng, Yanan Shi, Peng Sun PII:
S0024-3205(19)31118-X
DOI:
https://doi.org/10.1016/j.lfs.2019.117190
Reference:
LFS 117190
To appear in:
Life Sciences
Received date:
12 September 2019
Revised date:
4 November 2019
Accepted date:
14 December 2019
Please cite this article as: M. Li, J. Peng, Y. Shi, et al., miR-92a promotes progesterone resistance in endometriosis through PTEN/AKT pathway, Life Sciences(2019), https://doi.org/10.1016/j.lfs.2019.117190
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Journal Pre-proof 1 miR-92a Promotes Progesterone Resistance in Endometriosis Through PTEN/AKT Pathway Manchao Li§*, Jintao Peng§, Yanan Shi, Peng Sun The Department of Reproductive Medicine Center, The Sixth Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, China Both authors contributed equally to this work and should be considered as equal first
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coauthors.
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*Corresponding author
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Manchao Li
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The Department of Reproductive Medicine Center, The Sixth Affiliated Hospital of Sun Yat-Sen University, No. 26 Erheng Road, Yuancun Street, Tianhe District, Guangzhou
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510000, Guangdong, China
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Tel: +86-18312025840
Email: [email protected]
Authors email Manchao Li
email: [email protected]
Jintao Peng
email: [email protected]
Journal Pre-proof 2 email: [email protected]
Peng Sun
email: [email protected]
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Yanan Shi
Journal Pre-proof 3 Abstract The alteration of PTEN expression may be a vital part of the pathological and physiological mechanisms in infertility-related with endometriosis. However, the potential mechanisms underlying abnormal expression of PTEN and its role in progesterone-resistant endometriosis have not been thoroughly elucidated. In this study,
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our data showed the PTEN messenger RNA (mRNA) level and protein expression was reduced in progesterone-resistant endometriosis tissue and primary stomal cells. Low
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levels of PTEN in endometrial stromal cells led to higher cell proliferation and resistance
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to progesterone. In terms of PTEN suppression in progesterone-resistant endometriosis,
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the mRNA level of miR-92a was correlated negatively with PTEN level. Transfection of
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miR-92a mimic reduced PTEN expression and made the stromal cells more resistant to progesterone treatment. Inhibition of miR-92a by its antagomir had the opposite effects.
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Results of the luciferase reporter assay for the 3′-nontranslated region suggested that
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miR-92a directly modulated PTEN levels. Moreover, miR-92a inhibition by its antagomir enhanced the therapeutic effect of progesterone, which suppressed stromal cell
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proliferation, and reduced the formation of ectopic lesions in the mouse model of endometriosis. Hence, this study revealed that miR-92a contributed to the development of progesterone resistant endometriosis by suppression of PTEN expression, and modulation of miR-92a might be a potential medical method of treating endometriosis. Keywords: miR-92a; mRNA; PTEN; Endometriosis
Journal Pre-proof 4 Introduction Endometriosis is considered as a gynecological disorder, and defined as the growth of stroma and glands of the endometrium in extrauterine locations, mainly the rectovaginal septum, ovaries, and pelvic peritoneum (Bulun, 2009, Giudice, 2010). Endometriosis affects 6% to 10% of women in child-bearing age, and the incidence is 35% to 50% among those who suffer from chronic pelvic pain and infertility, which has a negative
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effect on the quality of life and on health (Moradi et al. , 2014). Current therapies include
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surgery, medicine, and combined treatment (Giudice, 2010). After surgical removal of
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endometriosis, 30% to 50% of patients experience disease recurrence in 3 to 5 years. The
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most surprising phenomenon is the reappearance of endometriosis in women with both
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uterus and ovaries excised (Bulun, 2009, Guo and Olive, 2007). In women of childbearing age, the therapy with gonadotropin-releasing hormone analogs, androgenic
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agents, progesterone, or oral contraceptives can be used only in the short term to maintain
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low estrogen levels, because of adverse effects, such as bone density loss and pseudomenopause (Bulun, 2009, Giudice, 2010, Guo and Olive, 2007). After low-
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estrogen therapy is discontinued, the rate of recurrence within a year is as high as 60% (Guo and Olive, 2007). Therefore, current therapies cannot meet the goal of reducing the reappearance of endometriosis and have an adverse influence on women’s reproductive health and pregnant ability. We thus investigated some underlying signal pathways related to nonsteroidal or nonestrogen targets for treating endometriosis. Recent studies of linkage and genomewide associations have revealed a strong connection between endometriosis and the 10q23-26, 7p15.2, and 9p21 loci. PTEN (phosphate and tension homolog deleted on chromosome 10) is a tumor-suppressing gene
Journal Pre-proof 5 with a double-specificity phosphatase at the 10q23 locus. Via a certain AKT-dependent pathway, this gene could repress cellular division and promote apoptosis (Sansal and Sellers, 2004). Immunohistochemistry assays demonstrated that the decrease in PTEN levels in normal endometrial glands (“PTEN-null glands”) appeared in 43% of samples from healthy women experiencing premature menopause. Follow-up tests after approximately 1 year demonstrated that PTEN-null glands could still be identified in 83%
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of the women (Mutter et al. , 2001). With mutation, deletion, or inactivation of PTEN, the
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expression of the PTEN protein is downregulated, and its activity is decreased. Therefore,
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the ability of PTEN protein to inhibit tumor cell proliferation, adhesion, migration, and
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angiogenesis is weakened, which thus promotes the development of tumors (Stahl et al. , 2003). Endometriosis, a largely benign, chronic inflammatory disease, is an independent
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risk factor for endometrioid and clear cell epithelial ovarian tumors. A previous study
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(Forster et al. , 2011) showed that the occurrence and development of endometrial diseases are closely related to the functional inactivation of PTEN. In endometriosis, it
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was demonstrated that the AKT pathway, in the downstream of PTEN, is overactive in
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comparison with endometrial cells from endometriosis-free patients (Yin et al. , 2012). However, the mechanism of PTEN protein inactivation in progestin treatment for endometriosis remains unclear. We hypothesized that low expression of PTEN protein also leads to progesterone resistance in endometriosis and that the pathogenesis of endometriosis might involve other potential mechanisms that modulate PTEN levels. Micro-RNAs (miRNAs) are small RNAs located in the noncoding areas of DNA that could prevent translation of or even degrade the target genes by linking to the 3′-untranslated region (UTR) in mRNAs
Journal Pre-proof 6 (Calin et al. , 2004). The modulation of PTEN is thought to be dependent on miRNA (Johnson et al. , 2005). In this study, we demonstrated that the low expression of PTEN is one of the sources of progesterone resistance in human endometriosis. The miR-92a is responsible for the upstream regulation of PTEN by targeting its 3′-UTR and contributes to progesterone resistance. Suppression of miR-92a can overcome progesterone resistance both in cells and in the whole organism by suppressing the proliferation
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process in stromal cells.
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Materials and Methods
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Tissue samples
The Sixth Affiliated Hospital of Sun Yat-Sen University Institutional Review Board
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approved our research. All participating patients were fully informed about the study and
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provided written consent to participate. During salpingo-oophorectomy or ovarian
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cystectomy, endometrial cyst walls were obtained from patients suffering from endometriosis in the Sixth Affiliated Hospital of Sun Yat-Sen University. All the patients had undergone progesterone therapy and were divided into two groups: those experienced pelvic pain relief after progesterone treatment (Responsive, n = 11), and those who did not have pelvic pain relief after progesterone treatment (Resistant, n = 12). Detailed information about the patients is listed in Table 1. The histological results of the samples were provided by pathologists.
Journal Pre-proof 7 Table
1.
The
information
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patients
endometriosis
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Stromal cell isolation and cell culture
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The stromal cells of the patients were isolated as described previously (Ryan et al. , 1994).
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In cell culture, we used Dulbecco’s modified Eagle’s medium (DMEM)/nutrient mixture
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F-12 in a ratio of 1:1 (Invitrogen, Carlsbad, Calif.) supplemented with fetal bovine serum
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(10%) and streptomycin, 100 U/mL, with penicillin, 100 U/mL, in an environment of 37°C with a wet atmosphere and CO2 (5%). Laboratory-grown human endometrial
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stromal cells (SHT290; Kerafast, Boston) were cultured in DMEM combined with
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epidermal growth factor (20 ng/mL; Becton Dickinson, Franklin Lakes, N.J.), glutamine
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(2 mM), HyClone fetal bovine serum (2%; Thermo Fisher Scientific, Waltham, Mass), and streptomycin, 100 U/mL, with penicillin, 100 U/mL, in a wet environment of 37°C supplemented with CO2 (5%). For both patients’ cells and the SHT290 cells, we changed the medium every 3 days. After the seventh pass, the cells were prepared for the tests described in the next sections. Specific concentrations of progesterone (Sigma-Aldrich, Shanghai) were used to treat cells. Cell viability and proliferation
Journal Pre-proof 8 Primary stromal cells or SHT290 cells were seeded in 96-well plates at a concentration of 5 × 104 cells in each well, and the cells were left overnight for attaching. Afterward, the cells were rinsed with phosphate buffer solution and then incubated in DMEM/F-12 (1:1; phenol red-free) supplemented with fetal bovine serum (10%) and streptomycin, 100 U/mL, with penicillin, 100 U/mL, and also progesterone in different concentrations for 24 hours. To detect cellular viability, we used the MTT assay kit (Sigma-Aldrich) in
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accordance with the manufacturer’s protocols, and we measured fold change in relation
incorporation
immunoassay
(Sigma-Aldrich)
in
accordance
with
the
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(BrdU)
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to vehicle control. To detect cell proliferation, we used the 5-bromo-2′-deoxyuridine
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manufacturer’s protocols, and we measured fold change in relation to vehicle control.
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Cell transfection and grouping
All the sequences we used were obtained from Shanghai Sangon Biological Engineering
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Technology & Services Co., Ltd. (Shanghai) as described in an earlier report (Li et al. ,
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2018). Before cell transfection, we cultured the cells in 6-well plates at a concentration of
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2 × 106 cells in each well. The confluence of the cells was 70% to 80% when the transfection was conducted. In accordance with the manufacturer’s protocols for Lipofectamine 2000 kits (Invitrogen), Gibco Opti-MEM medium without serum (250 μL; Thermo Fisher Scientific) was combined in four separate mixtures—with 100 pmol of miR-92a mimic, with PTEN small interfering RNAs (siRNAs)-, with miR-92a antagomir, and with normal control—with ultimate concentrations of 50 nmol/L. Afterward, the mixtures were cultured at normal room temperature for 5 minutes. Then, Gibco OptiMEM medium without serum (250 μL) was mixed with Lipofectamine 2000 (5 μL) and cultured at normal room temperature for 5 minutes. The two mixtures mentioned
Journal Pre-proof 9 previously were combined, cultured for 20 minutes at normal room temperature, and then added to the cells. After 6 hours of transfection, we replaced the mixtures with the complete medium and cultured the cells in a 37°C incubator with a wet atmosphere supplemented with CO2 (5% ) and room air for 24 to 48 hours in order to conduct the next experiments.
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Reverse transcription-quantitative polymerase chain reaction
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In accordance with the protocol for using TRIzol reagent (Invitrogen), we extracted total
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RNA from endometriosis tissues and cells so as to measure the purity and concentration of the RNA. The RNA we extracted was kept in a
80°C refrigerator for the next assays.
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In accordance with the protocols for the complementary DNA (cDNA) synthesis kit
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(TaqMan MicroRNA Reverse Transcription Kit; Thermo Fisher Scientific), we added the
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sterile water to dilute the cDNA we obtained from reverse transcription (diluting ratio, 1:5), and then preserved them in a 20°C refrigerator for next assays. The primers for
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PTEN and miR-92a were provided by Shanghai Biowing Applied Biotechnology Co.,
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Ltd. (Shanghai). The polymerase chain reaction (PCR) system (20 μL in total) included 0.4 μL of 50× Rox Reference Dye, 2 μL of cDNA (diluted 1:5), 2 μL of Universal Adaptor PCR Primer (2 μmol/L), 2 μL of All-in-One miRNA Q-PCR Primer (2 μmol/L), 20 μL of DNase/RNase-free H2O, and 10 μL of 2 × All-in-One Q-PCR Mix. The amplification conditions consisted of predenaturation at 95°C for 10 minutes; denaturation at 95°C for 40 cycles, each lasting 10 seconds; annealing at 60°C for 20 seconds; and the elongation process at 72°C for 34 seconds. We used the Applied Biosystems 7500 quantitative PCR system (Applied Biosystems, New York) to conduct reverse transcription-quantitative -PCR. The β-actin acted as an internal reference in the
Journal 10 Pre-proof detection of miR-92a in the measurement of PTEN. We used the 2−ΔΔCt method to measure the relative expression of the target genes. The primers sequences were as described in an earlier report (Li et al., 2018). Immunoblotting The patients’ stromal cells and the SHT290 cells were rinsed with ice-cold phosphate
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buffer solution, and we used M-PER Mammalian Protein Extraction Reagent (Thermo
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Fisher Scientific, Shanghai) with phosphatase inhibitors and protease (both from Sigma-
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Aldrich, St. Louis) on ice to obtain cell lysates. The Micro BCA Protein Assay Kit (Thermo Fisher Scientific) was used to determine the total protein concentrations.
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Proteins in equal amounts were separated on 10% polyacrylamide gel and then
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transferred onto polyvinyl difluoride membranes. Then, 5% bovine serum albumin in trisbuffered saline with 0.1% Tween 20 (TBST; Sigma-Aldrich) was used to block the
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membranes for 1 hour at room temperature. Afterward, the membranes were incubated
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for 1 night in a 4°C refrigerator with primary antibodies (in 1% bovine serum albumin)
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against β-actin (Sigma-Aldrich), Ki-67 (Cell Signaling Technology, Danvers, Mass), PTEN, total AKT, and phospho-AKT (Ser473). The next day, the membranes were washed with TBST and then incubated with anti-mouse or goat anti-rabbit secondary antibodies bound with horseradish peroxidase (concentration, 1:4000) for 1 hour. Then, with a kit for chemiluminescent detection (Thermo Fisher Scientific), a Fujifilm LAS3000 Imager (Fujifilm, Tokyo) was used to take digital pictures of the Western blots. Immunohistochemistry assay
Journal 11 Pre-proof Paraformaldehyde (4%) was used for tissue fixation, and then the tissues were embedded in paraffin. We cut the embedded tissues into 4-μm-thick sections and placed them onto the glass slides. The sections were then deparaffinized and rehydrated, and we performed antigen restoration by heating the sections in citrate buffer (pH, 6.0). By culturing the sections in 0.03% H2O2 for 10 minutes, we blocked the activity of endogenous peroxidase. Dako protein block (Agilent Technologies, Santa Clara, Calif.) was applied
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to the sections for 30 minutes, and then the slides were rinsed with TBST and cultured
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with the primary antibodies against Ki-67 (1:100; Cell Signaling Technology) overnight
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in a 4°C wet box. The next day, the slides were washed with TBST, and then we applied
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secondary antibodies conjugated with horseradish peroxidase for 30 minutes. Diaminobenzidine tetrahydrochloride was used as a substrate to detect horseradish
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Luciferase reporter assay
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peroxidase activity, and the slides were counterstained with hematoxylin.
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The dual-luciferase reporter experiment was conducted for further gene identification.
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We cloned and amplified the full length of the 3′-UTR of PTEN. We subcloned the PCR products and inserted them in the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega Corporation, Madison, Wisc.), and we named them “PTEN-wt” (PTENwide type). Afterward, we conducted mutagenesis directed by site in the combined site in miR-92a and in the target gene obtained from bioinformatics software so as to establish a carrier for the PTEN mutant (“PTEN-mut”). We used the pRL-TK carrier that expressed renilla luciferase (Takara Holdings Inc., Kyoto) as the internal control in order to eliminate the differences in efficiency and cell amounts of transfection. The miR-92a antagomir and mimics were separately transfected into SHT290 cells, together with
Journal 12 Pre-proof carriers of luciferase reporter, and the activity of the dual-luciferase reporter gene has been measured with the methods provided by Promega Corporation. Induction of endometriosis in mice Animals were kept in a specific room in which care was based on the instructions from The Sixth Affiliated Hospital of Sun Yat-Sen University's institution of the use and care
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for experimental animals. By placing endometrial tissue in the animals’ peritoneal cavity,
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we established the endometriotic lesions in vivo. Tribromoethanol (Avertin) injection
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was used to produce deep anesthetization in the animals. We made a 1-cm midventral incision for laparotomic access to the peritoneal cavity, identified the intestine and uterus,
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excised the left uterine horn, and placed it in Basal Medium Eagle (Invitrogen) in a petri
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dish. The mean weight of the samples was 56.70 ± 5.47 mg. We cut the uterine horn open along the long axis and cut it into small pieces. The pieces were resuspended in Basal
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Medium Eagle (0.5 mL) and placed back into the abdominal cavity in the same mouse
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whose uterus has been excised to perform the autologous implantation. Then we gently
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massaged the abdomen to make the tissues fully disperse. 17-Estradiol pellets (SE-121; Innovative Research of America, Sarasota, Fla.), 0.36 mg/day, were implanted subcutaneously into the mice for 60 days to promote the formation of lesions similar to those of endometriosis. Mice were treated with progesterone, 50 mg/kg, with or without anti-miR92a, 10 μM/kg subcutaneously, once every 4 days starting on the fourth day before lesion formation was induced. After 4 weeks, the experimental mice were sacrificed. We opened the abdominal cavity and then counted and excised the endometriosis-like lesions. Those lesions and the uterine tissues were fixed in paraformaldehyde (4% in phosphate buffer solution) for further histological analysis.
Journal 13 Pre-proof Statistics analysis SPSS 18.0 software (SPSS Inc., Chicago) was used for statistical analysis. Chi-square tests were used to compare data between groups. The measurements were recorded as means ± standard deviations. If the data displayed homogeneous variance and were regularly distributed, we conducted t-tests to compare the two groups. P values lower
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than 0.05 were considered statistically significant.
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Results
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PTEN level was suppressed in women with progesterone-resistant endometriosis
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To detect the expression of PTEN and discover whether resistance to progesterone was present in women with endometriosis, we conducted quantitative reverse transcription
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PCR to measure levels of PTEN mRNA, using the endometrial samples from 11 women
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with progesterone-responsive endometriosis and 12 women with progesterone-resistant
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endometriosis. The levels of PTEN mRNA were remarkably lower in progesteroneresistant endometriosis samples than in the samples of patients with endometriosis, which can be relieved by progesterone treatment (Figure 1A). Consistently, the results of the Western blot also demonstrated that the expression of PTEN was lower in the patients with progesterone-resistant endometriosis (Figure 1B). After isolating the primary stromal cells from the tissue in progesterone-responsive and progesterone-resistant patients, we found that the progesterone-resistant cells had higher proliferation ability than did the progesterone-responsive cells (Figure 1C). Progesterone treatment suppressed the proliferation of progesterone-responsive stromal cells but showed no
Journal 14 Pre-proof effect on the proliferation process of progesterone-resistant stromal cells (Figure 1C). In further confirmation of the results, the BrdU assay and Ki-67 Western blot produced similar results (Figure 1D, E). In the progesterone-resistant stromal cells, the expression of PTEN was suppressed, but AKT was hyperactivated (Figure 1E). Moreover, the induction of PTEN and suppression of AKT by progesterone in responsive stromal cells were also abolished in the resistant stromal cells (Figure 1E). Therefore, our data
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indicated that PTEN expression was lower in the women with progesterone-resistant
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endometriosis.
Figure 1. PTEN expression was lower in progesterone resistant endometriosis. (A) The messenger RNA level of PTEN in endometrial biopsy specimens from patients with progesterone-responsive endometriosis (n = 11) and those with progesterone-resistant endometriosis (n = 12). (B) Representative Western blot of PTEN in endometrial biopsy specimens from patients with progesterone-responsive and patients with progesterone-
Journal 15 Pre-proof resistant endometriosis. The primary cells derived from these patients were treated with progesterone, 1 μM, for 48 hours. The cell proliferation was analyzed with MTT tests (C) and 5-bromo-2′-deoxyuridine (BrdU) assay (D). (E) The level of p-AKT, PTEN, and Ki67 has been analyzed with Western blot. Each experiment was repeated 3 times. N,
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p>0.05; *, p<0.05; **, p<0.01.
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Abnormal expression of PTEN mediated the progesterone resistance in endometriosis
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To evaluate the function of PTEN in progesterone resistance, a human endometrial
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stromal cell line, SHT290, was used in the following studies. We found that the transfection of PTEN siRNA in SHT290 cells suppressed the expression of PTEN
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(Figure 2A) but increased the expression of Ki-67, as well as activation of AKT (Figure
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2A), which suggested the higher proliferation of stromal cells after PTEN knockdown. The MTT assay and BrdU analysis also indicated that the absence of PTEN promoted the
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proliferation of SHT290 cells (Figure 2B, C). The depletion of PTEN also ameliorated
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the ability of progesterone to inhibit the proliferation of SHT290 cells (Figure 2B, C). To further confirm the effect of PTEN, we treated the progesterone resistant primary stromal cells with AKT inhibitor, LY294002 because activation of AKT occurs downstream of PTEN loss. As predicted, LY294002 suppressed the proliferation ability of progesteroneresistant stromal cells and also increased progesterone resistance in stromal cells (Figure 2D, E). These findings suggested that dysregulation of PTEN contributed to progesterone resistance in endometriosis.
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Figure 2. Absence of PTEN contributed to progesterone resistant in endometriosis. (A) The levels of PTEN and Ki-67 in SHT290 cells that were transfected with PTEN siRNA.
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The SHT290 cells transfected with PTEN siRNA were treated with 1 μM progesterone.
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The proliferation of cells was analyzed with MTT assay (B) and 5-bromo-2′deoxyuridine (BrdU) assay (C). The primary stromal cells from patients with
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progesterone-resistant endometriosis were treated with progesterone, 1 μM, with or
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without the combination of LY294002, 1 μM. The proliferation of cells was analyzed with MTT tests (B) and BrdU assay (C). Each experiment was repeated 3 times. N, p>0.05; *, p<0.05; **, p<0.01.
Journal 17 Pre-proof An increased miR-92a level is related to downregulated PTEN expression in progesterone-resistant endometriosis MiRNA was reported to modulate PTEN expression (Li et al., 2018). To reveal the potential mechanism of low PTEN expression in progesterone-resistant endometriosis, we analyzed the expression of several miRNAs that were reported to target PTEN,
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including miR-92a (Li et al., 2018), miR-26a (Xiong et al. , 2019), miR-130b, miR-494 (Lu et al. , 2019), miR-103 (Jiang et al. , 2019), and miR-21 (Liu et al. , 2019). The
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results from quantitative reverse transcription PCR experiments indicated that the miR-
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92a level increased remarkably in the patients with progesterone-resistant endometriosis
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(Figure 3A). In contrast, no great difference has been shown by other miRNAs between
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progesterone-responsive and progesterone-resistant endometriosis. The expression of miR92a was also negatively related to the mRNA level of PTEN in the 23 patients in this
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study (Figure 3B). On the basis of our results, we performed further studies on cells to
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explore the potential mechanism in the opposite relation between miR-92a and PTEN levels. Similarly, we discovered that the level of miR-92a was also higher in the primary
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stromal cells derived from patients with progesterone-resistant endometriosis (Figure 3C). Therefore, our data suggested that miR-92a might be the upstream regulator of PTEN in progesterone-resistant endometriosis.
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Figure 3. The level of miR-92a has been increased in progesterone resistant
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endometriosis. (A) The messenger RNA level of micro-RNAs (miRNAs) in endometrial biopsy specimens from patients with progesterone-response endometriosis (n = 11) and patients with progesterone-resistant endometriosis (n = 12). (B) The correlation of miR92a level with the messenger RNA level of PTEN in patients with endometriosis (n = 23). (C) The miR-92a level in primary stromal cells from progesterone-responsive or progesterone-resistant patients. Each experiment was repeated 3 times. N, p>0.05; **, p<0.01.
Journal 19 Pre-proof miR-92a reduces the PTEN level in cells via aiming at the 3′-untranslated region To further verify that PTEN is the target for miR-92a in the downstream, we detected the PTEN mRNA level under miR-92a activation and inhibition. The transfection of stromal cells with miR-92a mimic dramatically increased the miR-92a level (Figure 4A). Conversely, transfection with miR-92a antagomir dramatically suppressed miR-92a expression (Figure 4A). Upon investigating the effect of miR-92a activation or inhibition
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on the mRNA level of PTEN, we found that overexpression of miR-92a suppressed
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PTEN expression, but inhibition of miR-92a could elevate the mRNA expression of
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PTEN (Figure 4B). To confirm PTEN as a target gene for miR-92a, we respectively
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inserted PTEN-mut and PTEN-wt sequences in the reporter plasmids. The miR-92a antagomir or mimics and PTEN-wt or PTEN-mut recombined plasmids were transfected
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together into the SHT290 cells with the luciferase reporter gene. Our results
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demonstrated that the miR-92a mimic suppressed the PTEN-wt luciferase activity, which was enhanced by miR-92a antagomir co-transfection (Figure 4C). However, the co-
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transfection of the miR-92a mimic or antagomir showed no remarkable influences on the
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activity of luciferase on PTEN-mut plasmids (Figure 4D). The miR-92a mimic also suppressed the protein level of PTEN, but its antagomir enhanced the expression of PTEN in SHT290 cells (Figure 4E). These findings indicate that PTEN could be one of the target genes for miR-92a.
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Figure 4. MiR-92a expression targeted the 3′-untranslated region (UTR) in PTEN. (A) The miR-92a level in SHT290 cells that were transfected by antagomir (right) or miR92a mimic (left). (B) The messenger RNA level of PTEN in SHT290 cells that were transfected by miR-92a antagomir or mimic. (C) The activity of PTEN-wide-type luciferase reporter gene in SHT290 cells that were transfected by miR-92a antagomir or mimic. (D) The activity of 3′-UTR mutant PTEN luciferase reporter gene in SHT290
Journal 21 Pre-proof cells that were transfected by miR-92a antagomir or mimic. (E) PTEN and phospho-AKT level in SHT290 cells that were transfected by miR-92a antagomir or mimic. Each experiment was repeated 3 times. N, p>0.05; **, p<0.01; ***, p<0.001.
MiR-92a overexpression suppresses the effect of progesterone in vitro
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To identify the relationship between miR-92a and PTEN levels in progesterone resistance,
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we studied the effects of this relationship on SHT290 cell proliferation. Transfection with
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miR-92a promoted the proliferation of SHT290 cells and made them resistant to
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progesterone (Figure 5A). Results of the BrdU assay also indicated that miR-92a overexpression promoted SHT290 cell proliferation (Figure 5B) and compromised the
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inhibitory effect of progesterone (Figure 5B). Conversely, transfection with miR-92a
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antagomir inhibited the proliferation of SHT290 cells and also enhanced the suppressive effects of progesterone (Figure 5C). The BrdU assay yielded similar results (Figure 5D).
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All these results suggested that miR-92a expression contributed to progesterone
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resistance in endometriosis.
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Figure 5. MiR-92a expression mediated the progesterone resistance in stromal cells. The
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SHT290 cells that were transfected by miR-92a mimic were treated with progesterone, 1 μM. The cell proliferation ability was analyzed with MTT tests (A) and 5-bromo-2′deoxyuridine (BrdU) assay (B). The SHT290 cells transfected with miR-92a antagomir were treated with progesterone, 1 μM. The cell proliferation was analyzed by MTT assay (C) and BrdU assay (D). Each experiment was repeated 3 times. N, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001.
Journal 23 Pre-proof Inhibition of miR-92a restored endometriosis to progesterone treatment in vivo To clarify the effect of miR-92a inhibition on progesterone resistance in endometriosis, we induced endometriosis in syngeneic mice. In these model animals, small endometrial cell groups without myometrium were placed in the peritoneum cavities of syngeneic female mice to help the cells randomly attach and grow. We excised the ovaries in those mice and then subcutaneously administered progesterone with or without anti-
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miR92a antagomir to these mice once every 4 days starting, on the fourth day before the
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induction of lesions. We discovered that miR-92a antagomir remarkably suppressed the
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miR-92a level (Figure 6A), which was suggestive of the efficiency of antagomir in
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animal models. The number and volume of the abnormal lesions were calculated in the mice with the dissecting microscope, and then we conducted histological evaluations. All
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transplanted endometriotic lesions were found in the peritoneum. The progesterone or
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antagomir single treatment did not significantly reduce the number or volume of lesions (Figure 6B). However, the combined treatment significantly suppressed the formation
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and volume of the ectopic lesions (Figure 6B). Western blot revealed that miR-92a
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antagomir enhanced the PTEN level but suppressed AKT activation (Figure 6C). Furthermore, the Ki-67 immunochemistry staining also indicated that miR-92a antagomir enhanced the inhibitory effect of progesterone on the proliferation of cells (Figure 6D). Therefore, our data collectively indicated that the administration of miR-92a antagomir can restore the progesterone response in endometriosis in vivo.
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Figure 6. Inhibition of miR-92a enhanced the therapy effect of progesterone in vivo. (A)
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The messenger RNA level of miR-92a in ectopic tissue of mice with endometriosis after injection with miR-92a antagomir. (B) Mice were treated with the administration of miR-
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92a antagomir, 10 mM/kg, or progesterone, 50 mg/kg, or both when tissue injection was
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conducted and then once every 4 days for 4 weeks (n = 6 for each group). Lesions and the relative volume in the peritoneal cavity were measured. (C) The level of AKT, phosphoAKT, and PTEN in the ectopic tissues from different groups of mice. (D) The Ki-67 staining of ectopic lesions. Each experiment was repeated 3 times. N, p>0.05; **, p<0.01; ***, p<0.001.
Journal 25 Pre-proof Discussion The typical characters of endometriosis are infertility and severe and cyclic chronic pain in the pelvis. The causes of endometriosis-related infertility still have not been defined well, but the disease is caused by abnormal expression of genes in normal endometrial tissue and also correlated with progesterone resistance (Bulun, 2009, Fazleabas, 2010, Giudice, 2010). Progesterone therapy in the form of regular hormonal treatments helps
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alleviate symptoms and inhibits the reappearance of endometriosis (Mounsey et al. ,
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2006). However, treatment efficacy is only suitable for a specific group of patients.
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Multiple models have revealed that PTEN plays an important role in the action of
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progesterone on the endometrium, and loss of PTEN expression hinders embryonic
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implantation in the endometriosis patients (Guzeloglu-Kayisli et al. , 2003, Kim et al. , 2014). However, whether abnormal expression of PTEN in endometriosis contributes to
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progesterone resistance is still unknown. We found that decreased expression of PTEN in
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endometriosis led to progesterone resistance. We also identified a novel miRNA, miR92a, which was associated with PTEN suppression in endometriosis. The results from the
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cell and animal experiments suggested that inhibiting miR-92a increased the expression of PTEN and overcame progesterone resistance by suppressing the stromal cell proliferation. Thus MiR-92a could represent a new therapy for endometriosis. PTEN is a tumor suppressor gene that acts against the AKT/phosphatidylinositol 3-kinase (PI3K) signal pathway, inhibiting cell proliferation and survival rate (Risinger et al. , 1997, Steck et al. , 1997). Earlier research suggested that changes in the PTEN gene frequently occur in tissues from the endometrium of women who suffer from endometrial hyperplasia and endometriosis, as well as ovarian and endometrial tumors (Castiblanco et
Journal 26 Pre-proof al. , 2006, Dinulescu et al. , 2005, Mandai et al. , 2009). In ovaries from both humans and mice, the PI3K-PTEN-AKT-Foxo3 pathway was activated in samples from women with endometriosis. Administration of AS101 restored the proportion of primordial follicles in the ovaries of mice with endometriosis to control levels (Takeuchi et al. , 2019). In our study, we found that human patients with endometriosis, who had low PTEN expression, developed progesterone resistance. It was reported that progesterone treatment
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upregulated the expression of PTEN to suppress endometriosis proliferation (Mutter et
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al. , 2000). Our in vitro data also indicated that silence of PTEN in stromal cells enhanced
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cell proliferation and abolished the suppressive effect of progesterone (Fig. 2). Therefore,
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it is reasonable that low expression of PTEN results in progesterone resistance in
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endometriosis.
More importantly, accumulating evidence suggested that ovarian and endometrial tumors
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are usually identified in relation to endometriosis, which indicates that they represent
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malignant transformation (Dinulescu et al., 2005, McMenamin et al. , 1999). Thus women with endometriosis may have increased risks for multiple cancers of various
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kinds (Verri et al. , 2005). Hence, complete loss of or mutations in PTEN might result in the formation and growth of endometriosis, which might eventually turn into cancer. Our results about targeting miR-92a to reverse PTEN expression not only provided a new concept to overcome the progesterone resistance in endometriosis but also can protect patients with endometriosis from malignant transformation. Over a long time, many researchers have investigated the probable function of miRNA in endometriosis formation (Grechukhina et al. , 2012, Pei et al. , 2018). In this study, we noticed a remarkable elevation of the miR-92a level in progesterone-resistant
Journal 27 Pre-proof endometriosis, which was correlated with the suppression of PTEN expression. According to our data, the cells transfected with miR-92a mimics demonstrated decreased PTEN levels but enhanced AKT phosphorylation process. Some researchers have shown that miR-92a had a close association with human malignancy (Li et al., 2018). In gastric malignancy, miR-92a is considered oncogenic, with an important role in promoting cell proliferation by suppressing FBXW7, a tumor-suppressing gene (Liu et al. , 2016). In
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contrast, inhibiting miR-92a could dramatically impede cell growth and promote cell
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apoptosis, which has been studied in research (Sharifi et al. , 2014). A reduced PTEN
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level and an elevated miR-92a level were discovered in tissues from colorectal cancer,
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which implicated that they were negatively correlated (Ke et al. , 2015). Furthermore, miR-92a facilitates tumor development and inhibits immune activity by activating the
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MAPK/ERK signal pathway through the suppression of PTEN in cervical malignancy (Li
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et al., 2018). However, the expression of miR-92a in women who suffer from endometriosis is still unknown. In this study, we initially demonstrated that the
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proliferation of endometrial stromal cells was enhanced after the transfection of anti-
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PTEN siRNA or miR-92a mimic. Accordingly, our data revealed that the high expression of miR-92a suppresses the expression of PTEN, which results in progesterone resistance in endometriosis. These data revealed a novel function of miR-92a in endometriosis.
In conclusion, this research suggests that miR-92a is highly expressed in the progesterone-resistant endometriosis, which suppresses the expression of PTEN. Our findings reveal that the efficiency of progesterone in endometriosis therapy could be poor as a result of downregulated PTEN signal by miR-92a dysregulation, which suggests a potential application of miRNAs as biological therapy in the treatment of endometriosis.
Journal 28 Pre-proof Acknowledgements: None.
Conflicts of interest: The authors confirm that there are no conflicts of interest.
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Funding: This research did not receive any specific grant from funding agencies in the
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public, commercial, or not-for-profit sectors.
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Author contributions
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Study design/planning: Manchao Li, Jintao Peng
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Data collection/entry: Manchao Li, Jintao Peng
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Data analysis/statistics: Manchao Li, Jintao Peng, Yanan Shi, Peng Sun
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Data interpretation: Manchao Li, Jintao Peng, Yanan Shi, Peng Sun Preparation of manuscript: Manchao Li, Jintao Peng Literature analysis/search: Yanan Shi, Peng Sun
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