Abstract STUDY QUESTION Is it possible to improve fibrosis in endometriosis by microRNA-214 delivery in exosomes? SUMMARY ANSWER Upregulation of miR-214 may inhibit fibrogenesis and its delivery by exosomes derived from ectopic endometrial stromal cells (ESCs), offers an alternative therapeutic approach for endometriosis fibrosis. WHAT IS KNOWN ALREADY Fibrosis is the primary pathological feature of endometriosis. MiR-214 plays an important role in fibrotic disease. Connective tissue growth factor (CTGF) is a critical fibrogenic mediator of miR-214. The expression of miR-214 is decreased in ectopic ESCs compared with normal ESCs. miRNAs are a natural cargo of exosomes and these could be exploited as carriers of miRNA in replacement therapy. STUDY DESIGN, SIZE, DURATION Paired eutopic and ectopic endometrial tissue samples were obtained from 10 women with ovarian endometrioma. ESCs and epithelial cells from both were cultured in vitro. RT-PCR, western blot and immunohistochemistry were used to study the effect of transfection with miR-214 mimics on CTGF expression and fibrogenesis respectively, with and without TGFβ stimulation. Exosomes were isolated from ectopic ESCs and Endometrioma tissue was isolated from four patients, dispersed an injected (ip) into nude mice and allowed to implant. The mice were treated with miR-214-enriched exosomes or controls to confirm the effect of inhibiting CTGF overexpression on endometriosis fibrosis. PARTICIPANTS/MATERIALS, SETTING, METHODS The primary ectopic ESCs were transfected with miR-214 mimics. The levels of miR-214, CTGF and fibrotic markers were measured by RT-PCR and Immunohistochemistry. A mouse model of endometriosis was established by ip injection of human ectopic endometrial tissues into nude mice. MiR-214-enriched exosomes were injected into the mice and endometriotic lesions were measured on Day 28. Changes in fibrosis of the endometriotic implants were studied by histopathological staining. MAIN RESULTS AND THE ROLE OF CHANCE CTGF and fibrotic markers upregulation in endometriosis is associated with a reciprocal down-regulation of miR-214. By using miR-214 mimics and antagomirs to investigate expression of fibrotic markers, we found that increased production of miR-214 reduced Collagen αI and CTGF expression in endometriosis stromal and endometrial epithelial cells in response to fibrosis-inducing stimuli (P < 0.001 versus non-treatment). Ectopic ESCs yielded nano-sized exosomes which expressed miR-214. Loading exosomes with miR-214 mimics and injecting them into an experimental endometriosis mouse model resulted in a decrease in the expression of fibrosis-associated proteins (P < 0.001 versus PBS control group). LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION We only isolated exosomes from ectopic ESCs, whether this is the optimum source requires further study. WIDER IMPLICATIONS OF THE FINDINGS Upregulation of miRNA-214 potentially offers an alternative therapeutic approach for endometriosis fibrosis. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by grants from the National Natural Science Foundation of China (Grant no. 81771549 Jinwei Miao). The authors declare that there is no conflict of interest. endometriosis, fibrosis, miR-214, CTGF, exosome Introduction Endometriosis is a chronic gynecology disorder characterized by the development of endometrial tissue outside of the uterus, mainly implanted over the visceral and peritoneal surfaces in the female pelvis. Endometriosis is often accompanied by chronic pelvic pain and infertility. Estimates suggest that up to 80% of women with chronic pelvic pain may be affected by endometriosis and that 20–50% of women being treated for infertility have endometriosis (Mishra et al., 2015; Sabuszewska-Jwiak et al., 2015). Although the pathogenesis of endometriosis remains elusive, fibrosis is the primary pathological feature of endometriosis characterized by excessive deposition and reorganization of extracellular matrix (ECM) during the development and progression of endometriotic tissues (Matsuzaki and Darcha, 2014; Malutan et al., 2015). Histologically, dense fibrous tissue is seen surrounding the endometrial glands and stroma in endometriosis (Malutan et al., 2015). Excess fibrosis may lead to scarring, altered tissue function and resistance to hormonal suppressive therapy (Matsuzaki et al., 2010). Novel therapies targeting the mechanisms of fibrosis in endometriosis are indispensable for the development of strategies to prevent and treat endometriosis. Transforming growth factor-β (TGF-β) is up-regulated and activated in fibrotic diseases and modulates fibroblast phenotype and function, inducing myofibroblast transdifferentiation while promoting matrix preservation. However, the pleiotropic and multifunctional effects of TGF-β raise concerns regarding potential side effects that may be caused by TGF-β blockade. Some of the pro-fibrotic effects of TGF-β are mediated through upregulation of its downstream effector connective tissue growth factor (CTGF, also known as CCN2). CTGF can also bind directly to TGF-β, and enhance its activity resulting in increased binding to TβRI and TβRII (Biernacka et al., 2011). In addition, increased expression of CTGF was observed in endometrium with fibrosis and/or inflammation (Rebordão et al., 2014). Therefore, CTGF is an attractive therapeutic target as supported by the broad anti-fibrotic efficacy of CTGF antagonists in vitro or in experimental fibrosis models in vivo (Katsunari et al., 2017). MicroRNAs (miRNAs), a sort of non-coding RNA, play a role in regulating gene expression at the post-transcriptional level by binding to single or multiple 7–8 mermotifs in the 3′-untranslated regions (3′-UTRs) of the target mRNAs (Bartel, 2004). A single miRNA can target dozens of genes, resulting in the adjustment of the target mRNA expressions (Pillai, 2005; Engels and Hutvagner, 2006). Previous microarray studies have detected some miRNAs that are aberrantly expressed in endometriosis (Teague et al., 2010; Braza-Boïls et al., 2014). MiR-214, one of the miRNAs down-regulated in human endometriotic cyst stromal cells (ECSCs) (Abe et al., 2013), is known to have fibrosis-suppressor roles, including the inhibition of fibroblast proliferation, collagen synthesis and the epithelial–mesenchymal transition process (Lv et al., 2017). CTGF is a predicted target molecule of miR-214 (Chen et al., 2015). Chen et al. (2014) found that miR-214 could inhibit fibrogenesis in hepatic stellate cells by targeting CTGF. However, it remains unclear whether the miR-214 involved in the pathogenesis of endometriosis fibrosis targets CTGF. Here, we test the hypothesis that miR-214 expression is reduced in fibrotic endometriosis and aberrant epigenetic regulation of CTGF may mediate the mechanisms of fibrogenesis in endometriosis. Exosomes are nano-sized membranous vesicles that are secreted by a variety of cell types and tissues (Harp et al., 2016). Recent evidence indicates that exosomes are natural carriers of miRNAs and could be exploited as such in miRNA replacement therapy (Zhou et al., 2016; Letelier et al., 2016). In this study, we aimed to evaluate whether exosomes loaded with miR-214 could reverse fibrosis by targeting CTGF in a xenograft model of endometriosis using immunodeficient nude mice. Materials and Methods Patients and tissue specimens Overall, 24 patients, aged 20–40 years, undergoing laparoscopy for ovarian endometriotic cysts at Beijing Obstetrics and Gynecology Hospital, Capital Medical University were recruited for the present study. After informed consent, paired eutopic and ectopic endometrial tissue samples were obtained from them. All patients had regular menstrual cycles and had ovarian endometriomas (rASRM Stages III–IV). None of the women had received hormonal treatments, such as gonadotropin-releasing hormone agonists (GnRHa) or steroid medication, and none had used intrauterine contraception for at least 6 months prior to surgery. As control samples, normal endometrial tissues were obtained from patients with uterine myomas who underwent laparoscopic myomectomy (n = 8, aged 25–50 years). Clinical samples used in this study were approved by Beijing Obstetrics and Gynecology Hospital affliated Capital Medical University Ethics Committee and written informed consents were obtained from all participants. Samples of endometrial and endometriotic tissue were divided into two portions. The first tissue portion was fixed in formalin and embedded in paraffin. The second portion was immediately placed in Hanks’ balanced salt solution (Gibco-BRL, Gaithersburg, MD, USA). Cell culture Paired eutopic and ectopic endometrial tissue samples were obtained from 10 patients with endometrioma. The tissue samples were minced into smaller pieces and incubated in Dulbecco’s modified Eagle’s Medium (DMEM)/F12 (Gibco-BRL) including 1% (v/v) penicillin/streptomycin, collagenase (1 mg/mL, 15 U/mg) and deoxyribonuclease (0.1 mg/mL, 1 500 U/mg) for 60 min at 37°C. The cells were pelleted, washed, suspended in DMEM/F12 containing 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin. Subsequently, the cells were filtered through a 40-μm cell strainer (BD, Le Pont de Claix, France). Endometrial epithelial cells (EECs) were collected from the pellet in sieves and suspended in the DMEM/F12 with 10% (v/v) FBS. The filtrate was centrifuged at 1000 g for 10 min at 4°C and the supernatant was discarded. Then eutopic (euto) and ectopic (ecto) endometrial stromal cells (ESCs) were suspended in the DMEM/F12 with 10% (v/v) FBS. Immunofluorescent staining was performed to determine the purity of the isolated EECs, euto-ESCs and ecto-ESCs using monoclonal antibodies for human cytokeratin (MNF116, 1:100, DAKO, Glostrup, Denmark), vimentin (V9, 1:100, DAKO), factor VIII (1:100, DAKO), as previously described (Matsuzaki and Darcha, 2013). All cells seeded in culture flasks and cultured at 37°C in an incubator under 5% CO2 in air. Cultured cells at 3–5 passages were used for further analysis. Treatment of primary cells Euto-ESCs, Ecto-ESCs and EECs were seeded into 24-well plates (5 × 104 cells per well) for quantitative real-time PCR and immunocytochemistry or 60-mm dishes for TGF-β1 treatment in culture media. At 80% confluency, TGF-β1 was added at concentrations of 0, 1, 2, 5 and 10 ng/mL (R&D System, Lille, France) and cultured for 24 h. Transfection of miRNA At 24 h prior to transfection, Euto-ESCs, Ecto-ESCs and EECs were seeded onto 6-well plates at 70–90% confluency. The cells were transfected with 100 nM miR-214 mimics or antagomirs (Invitrogen, Carlsbad, CA, USA) by mixing with Lipofectamine 2000 (Invitrogen) and OPTI-MEM for 24 h, according to the manufacturer’s instructions. Subsequently, the media were refreshed, and cells were stimulated with TGF-β1 at concentrations of 0 and 5 ng/mL (based on the results of the above experiment) for 24 h to investigate whether miR-214 could decrease TGF-β1 stimulated expression of fibrotic markers in euto-ESCs, ecto-ESCs and EECs. RNA extraction and RT-PCR To assess miRNA expression levels, RNA was extracted from cultured cells and tissues using microRNeasy mini kit (Qiagen, Valencia, CA, USA) and reversed transcribed using a miScript II RT kit (Qiagen) according to the manufacturers’ protocols. Quantitative real-time PCR for miRNAs was performed using SYBR Green Master Mix (Life Technologies) to assess the expression of miR-214 and glyceraldehyde-3-phosphatedehydrogenase (GAPDH). To assess mRNA concentrations, RNA was extracted from cultured cells and tissues by TRIzol Reagent (Invitrogen). Quantitative RT-PCR (qRT-PCR) was performed to detect CTGF, Collagen αI and GAPDH using a SYBR Green Master Mix (Life Technologies). All real-time PCR reactions were conducted in triplicate with an ABI PRISM 7000 Sequence Detection System. Primers used are shown in Supplementary Table SI. Western blot analyses Equal amounts of protein from normal endometrial tissues (n = 2) and ectopic endometrial tissues (n = 2) were resolved by 10% SDSPAGE (100 μg/lane) and transferred to PVDF membranes. The protein concentrations of the supernatants were determined using a Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA) with BSA as the standard. The membranes were blocked with non-fat milk for 1 h at room temperature, and then were incubated overnight with the primary antibodies: anti-CTGF (1:500; Abcam, Cambridge, MA, USA), anti-αSMA (1:100, Dako Cytomatio, Denmark), anti-collagen αI (1:250, Abcam) or anti-b-actin (1:500, Abcam) at 4°C for 2 h. Subsequently, suitable secondary antibodies were applied. All Western blotting assays were independently repeated for at least three times. In-situ hybridization (ISH) Fixed normal and eutopic endometrial sections were hybridized with miR-214 probes (Exiqon Inc., Woburn, MA, USA) (5′-ACTGCCTGTCTGTGCCTGCTG-3′) for 60 min at 55°C and then were washed with different concentrations ofsaline sodium citrate buffer. Slides were incubated with a monoclonal anti-digoxigenin-alkaline Phosphatase antibody (1:800) (Roche, Indianapolis, IN) for 60 min to detect probes, and by nitro-blue tetrazolium and 5-bromo-4-chloro-3′-indolyphosphate substrates (Roche) at 30°C for 2 h. The slides were mounted with Eukitt® Medium (VWR, Radnor, PA) and examined by confocal microscopy. Immunohistochemistry Ecto-ESCs and Euto-EECs were fixed in 2% (w/v) paraformaldehyde, permeabilized with 0.1% (v/v) Triton X-100 and blocked at room temperature. Human ectopic endometrial tissues and normal endometrial tissues fixed in a 4% (w/v) neutral paraformaldehyde solution, were waxed and embedded. Then sections (4 μm) were deparaffinized. All fixed endometrial sections or primary passaged cells were incubated with anti-CTGF (1:500, Abcam), anti- αSMA (1:100, Dako Cytomatio), or anti-collagen αI (1:250, Abcam) followed by Alexa Fluor® 488 goat-anti mouse IgG or Alexa Fluor® 568 goat-anti rabbit IgG (all at 1:1000; Life Technologies, Carlsbad, CA, USA) for 1 h at room temperature. The cells and coverslips were mounted by using Vectashield with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA), and examined by confocal microscopy. Isolation of exosomes Prior to exosome isolation, Ecto-ESCs were starved for 48 h. After incubation, the culture medium was collected and centrifuged at 300 g for 10 min to remove whole cells. The supernatant was subjected to a second centrifugation at 2000 g for 10 min to remove dead cells and then centrifuged again at 10 000 g for 30 min to remove large cell debris. After passing the supernatant through a 0.22-μm syringe filter (Millex, Millipore, Ireland), exosomes were pelleted by ultracentrifugation at 100 000 g for 70 min at 4°C. The pellet was resuspended in PBS. Exosome quantity was estimated by BCA assay. Characterization of exosomes from ecto-ESCs MiR-214 transfected or non-transfected ecto-ESCs were cultured in DMEM/F12 medium without serum for 48 h. Exosomal or cellular RNAs were isolated and the presence of miR-214 was determined by RT-PCR and its concentration normalized to total cellular GAPDH as described above. Purified exosomes were loaded on carbon-coated 400-mesh copper grids (Electron Microscopy Sciences, Hatfield, PA), stained with 2% uranyl acetate, air-dried, and imaged by transmission electron microscopy (TEM) with JEM-1400 plus at the Chinese Academy of Medical Sciences (CAMS). Purified exosomes were analyzed by dynamic light scattering using a BI 200SM Research Goniometer System (Brookhaven Instruments Inc., Holtville, NY). Zeta potential was determined with a ZetaPALS analyzer (Brookhaven Instruments Inc.). Experimental endometriosis model in Balb/c mice Animals Animal protocols were approved by the Committee on Ethical Use of Animals of Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Mice were maintained in a barrier unit in a well-controlled, pathogen-free environment with regulated cycles of light/dark (12 h/12 h, 23–25°C) and allowed a 2-week period of acclimation to the vivarium before any procedures were performed. The nude mouse model of endometriosis was used as previously described (Matsuzaki and Darcha, 2013). Briefly, 12 female nude mice (Balb/c, 6–8 weeks) were divided randomly into three groups (4 mice per group). Human eutopic endometrial tissue was recovered from four patients with endometriosis. For each patient endometrial fragments were suspended in PBS and passed through a 19-gauge needle before being injected (i.p.) into one anesthetized mouse from each group (~40 mg tissue/0.2 mL PBS per mouse, total three mice per patient). After the endometrial tissues had implanted for 14 days, one group of mice received miR-214 loaded exosomes derived from transfected ecto-ESCs-derived (~100 μg based on the results of preliminary experiment) by i.p. injection every 2 days until Day 14. Mice in the two control groups received PBS or exosomes from non-transfected ecto-ESCs. Over the experimental period, all mice survived, and there was no significant difference in growth rates between treated and untreated control mice. Mice were sacrificed 24 h after the last injection and the endometriotic implants were harvested for histology or RNA analysis as described above (Hsu et al., 2014). Histology Ectopic endometrial lesions from mice were fixed with 4% (w/v) paraformaldehyde for 24 h and then embedded in paraffin. Sections of 4 μm thickness were cut for histopathological examination. Paraffin-embedded tissue sections were stained with Masson Trichrome and Sirius Red to detect collagen fibrils that are deposited in the matrix, according to common protocols (Matsuzaki and Darcha, 2013). Results Endometriosis is associated with elevated expression of fibrotic markers and decreased expression of miR-214 To evaluate the expression of miR-214 in endometriosis, patients with (n = 6) or without (n = 6) endometriosis were recruited. We used in situ hybridization to detect miR-214 transcripts in ectopic endometrium from endometriosis patients and normal endometrium from control patients without endometriosis (Fig. 1A). It revealed less miR-214 expression in ovarian endometriosis compared with control patients. We next used real-time PCR to analyze the level of miR-214 and CTGF in ectopic and normal endometrium. The results showed that high levels of miR-214 and low levels of CTGF in normal endometrium compared with ectopic endometrium (Fig. 1B). Figure 1 View largeDownload slide Expression of miR-214 and fibrotic markers in normal endometrium and ectopic endometrium. (A) In situ hybridization analyses using DNA probe complementary to miR-214 performed on 5 μm sections of the tissue of ectopic endometrium and normal control. In situ hybridization shows that miR-214 (purple) is less expressed in ectopic endometrial tissues compare to normal endometrium. Scale bars=50 μm. (B) Expression of CTGF mRNA or miR-214 assessed by RT-PCR and normalized to glyceraldehyde-3-phosphatedehydrogenase (GAPDH) in normal endometrium and ectopic endometrium (n = 2 independent experiments performed in triplicate, *P < 0.001, +P < 0.05). (C) Western blotting indicated that CTGF, Collagen αI and α SMA proteins expression were decreased in ectopic endometrium. (D) Immunohistochemical detection of CTGF, α SMA or collagen αI in normal endometrium and ectopic endometrium. Specimens were also stained with 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain (blue). Scale bar = 50 μm. Figure 1 View largeDownload slide Expression of miR-214 and fibrotic markers in normal endometrium and ectopic endometrium. (A) In situ hybridization analyses using DNA probe complementary to miR-214 performed on 5 μm sections of the tissue of ectopic endometrium and normal control. In situ hybridization shows that miR-214 (purple) is less expressed in ectopic endometrial tissues compare to normal endometrium. Scale bars=50 μm. (B) Expression of CTGF mRNA or miR-214 assessed by RT-PCR and normalized to glyceraldehyde-3-phosphatedehydrogenase (GAPDH) in normal endometrium and ectopic endometrium (n = 2 independent experiments performed in triplicate, *P < 0.001, +P < 0.05). (C) Western blotting indicated that CTGF, Collagen αI and α SMA proteins expression were decreased in ectopic endometrium. (D) Immunohistochemical detection of CTGF, α SMA or collagen αI in normal endometrium and ectopic endometrium. Specimens were also stained with 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain (blue). Scale bar = 50 μm. As shown in Fig. 1C, the levels of CTGF, αSMA and Collagen αI proteins were significantly increased in ectopic endometrium compared with normal endometrium. The expression of αSMA and Collagen αI provides evidence of fibrosis. We also demonstrated that elevated levels of CTGF and fibrotic markers (αSMA and Collagen αI) were present in ectopic endometrial tissues (Fig. 1D) by immunohistochemistry. Statistics on fluorescent intensity from immunofluorescence images is shown in Supplementary Fig. S1A. Taken together, we found that miR-214 is down-regulated in patients with endometriosis, whereas CTGF and fibrotic markers are up-regulated. Effects of miR-214 on αSMA, collagen αI and CTGF In the study, we found a significant increase in the expression of Collagen αI and CTGF in euto-ESCs and ecto-ESCs in the presence of 5 ng TGF-β1/ml (Fig. 2A), and therefore 5ng/ml TGF-β1 was used to study the effect of miR-214. To demonstrate the functional link between CTGF, Collagen αI and miR-214, we transfected euto-ESCs and euto-ESCs with miR-214 mimics and successfully enhanced miR-214 levels (Fig. 2B). Treatment with miR-214 mimics significantly decreased the expression of αSMA, Collagen αI and CTGF mRNAs in both euto-ESCs and ecto-ESCs (Fig. 2C). In addition, as shown in Fig. 2C, TGF-β1 stimulation increased the expression of CTGF and fibrotic markers, and this effect was significantly attenuated by miR-214 in ecto-ESCs and euto-ESCs. MiR-214 is a product of dynamin 3 opposite strand (DNM3os) that directly suppresses CTGF mRNA (Chen et al., 2015). To determine the role of miR-214 in ESCs, ecto-ESCs and euto-ESCs were treated with antagomir. It revealed that the ability of miR-214 to inhibit CTGF and Collagen αI mRNA expression could be blocked by suppressing miR-214 levels (Fig. 2C). Immunofluorescence staining showed that the production of CTGF and Collagen αI protein were reduced when ecto-ESCs were transfected with miR-214 mimics (Fig. 2D), which is consistent with the data showing that the expression of fibrosis markers such as CTGF and Collagen αI are miR-214-dependent. Statistics on fluorescent intensity from immunofluorescence images is shown in Supplementary Fig. S1B Figure 2 View largeDownload slide Effects of miR-214 on αSMA, Collagen αI and CTGF. (A) Cultured ectopic endometrial stromal cells (ecto-ESCs) and eutopic endometrial stromal cells (euto-ESCs) were serum-starved for 24 h prior to treatment with 0–10 ng/ml TGF-β1. Subsequently, RNA was subjected to real-time PCR and expression of CTGF was normalized to that of GAPDH (n = 3 independent experiments performed in triplicate, *P < 0.001). (B) Relative expression of miR-214 in ecto-ESCs transfected with miR-214 mimics and its negative control (n = 3 independent experiments performed in triplicate, *P < 0.001). (C) Ecto-ESCs and euto-ESCs were transfected for 24 h with miR-214 mimics, some cells co-transfected with miR-214 antagomirs, serum starved for 24 h, and then treated with 5 ng/ml TGF-β1. Then cells were processed for real-time PCR of CTGF or Collagen αI expression (n = 5 independent experiments performed in triplicate, *P < 0.001). (D) ecto-ESCs and mir-214-transfected ecto-ESCs were evaluated for immunocytochemical detection of CTGF (red), collagen αI (green). Blue depicts DAPI nuclear stain. Scale bar: 20 μm. (E) Cultured EECs were serum-starved for 24 h prior to 48 h treatment with 0–10 ng/ml TGF-β1. Detection of CTGF (red) or Collagen αI (green) was shown by immunofluorescence. Scale bar: 20 μm. (F) Expression of CTGF mRNA or miR-214 assessed by RT-PCR and normalized to GAPDH (n = 3 independent experiments performed in triplicate, *P < 0.001). Figure 2 View largeDownload slide Effects of miR-214 on αSMA, Collagen αI and CTGF. (A) Cultured ectopic endometrial stromal cells (ecto-ESCs) and eutopic endometrial stromal cells (euto-ESCs) were serum-starved for 24 h prior to treatment with 0–10 ng/ml TGF-β1. Subsequently, RNA was subjected to real-time PCR and expression of CTGF was normalized to that of GAPDH (n = 3 independent experiments performed in triplicate, *P < 0.001). (B) Relative expression of miR-214 in ecto-ESCs transfected with miR-214 mimics and its negative control (n = 3 independent experiments performed in triplicate, *P < 0.001). (C) Ecto-ESCs and euto-ESCs were transfected for 24 h with miR-214 mimics, some cells co-transfected with miR-214 antagomirs, serum starved for 24 h, and then treated with 5 ng/ml TGF-β1. Then cells were processed for real-time PCR of CTGF or Collagen αI expression (n = 5 independent experiments performed in triplicate, *P < 0.001). (D) ecto-ESCs and mir-214-transfected ecto-ESCs were evaluated for immunocytochemical detection of CTGF (red), collagen αI (green). Blue depicts DAPI nuclear stain. Scale bar: 20 μm. (E) Cultured EECs were serum-starved for 24 h prior to 48 h treatment with 0–10 ng/ml TGF-β1. Detection of CTGF (red) or Collagen αI (green) was shown by immunofluorescence. Scale bar: 20 μm. (F) Expression of CTGF mRNA or miR-214 assessed by RT-PCR and normalized to GAPDH (n = 3 independent experiments performed in triplicate, *P < 0.001). EECs were CTGF and collagen αI positive in response to concomitant increases of TGF-β1 (Fig. 2E). Statistics on fluorescent intensity from immunofluorescence images is shown in Supplementary Fig. S1C. Similarly, EECs with TGF-β1 treatment expressed highly up-regulated CTGF and Collagen αI levels as assessed by RT-PCR (Fig. 2F). Exosomes characterization The exosomes-like vesicles were collected and purified by ultracentrifugation methods from conditioned medium from the ecto-ESCs culture. TEM confirmed the appearance of exosomes as 50–100 nm bi-membrane vesicles (Fig. 3A). Moreover, ecto-ESCs-derived exosomes with high negative charge (−20.1 mV) and diameter of ~65 nm were assessed by zeta potential analysis and dynamic light scattering respectively (Fig. 3B). Figure 3 View largeDownload slide Exosomes were isolated by sequential centrifugation of conditioned medium from ecto-ESCs. (A) Transmission electron microscopy (TEM). Scale bar=100 nm. (B) Dynamic light scattering analysis and zeta potential analysis, showing the presence of ~65 nm particles (−20.1 mV). (C) RT-PCR of cellular or exosomal miR-214 isolated from control or miR-214-transfected cells after 48 h, normalized to cellular GAPDH (n = 3 independent experiments performed in triplicate, *P < 0.001 versus control group). Figure 3 View largeDownload slide Exosomes were isolated by sequential centrifugation of conditioned medium from ecto-ESCs. (A) Transmission electron microscopy (TEM). Scale bar=100 nm. (B) Dynamic light scattering analysis and zeta potential analysis, showing the presence of ~65 nm particles (−20.1 mV). (C) RT-PCR of cellular or exosomal miR-214 isolated from control or miR-214-transfected cells after 48 h, normalized to cellular GAPDH (n = 3 independent experiments performed in triplicate, *P < 0.001 versus control group). Despite the relatively low abundance of miR-214 produced by the stromal cells, real-time PCR demonstrated that miR-214 was detectable in the exosomes from ecto-ESCs (Fig. 3C). In addition, compared with non-transfected ecto-ESCs-derived exosome, the exosomal concentration of miR-214 was significantly increased (~10-fold) in ecto-ESCs after 48 h of transfection (Fig. 3C). These exosomes were collected and used in the administration of mice with endometriosis. Mouse model of endometriosis Our in vitro experiments demonstrated that miR-214 suppresses the expression of CTGF and fibrosis markers (Collagen αI and αSMA). We next asked whether miR-214-enriched exosomes could affect endometriosis fibrosis in vivo. To test this hypothesis, we first established a mouse model of endometriosis. Fourteen days after intraperitoneal implantation, mice that received injections of endometrial fragments had endometrial-like lesions in the intestine, mesentery and peritoneum. In addition, adhesions were detected surrounding the endometriotic implants (Fig. 4A). As assessed by RT-PCR, we found that miR-214 levels were significantly elevated in endometriotic lesions of nude mice treated with 100 μg/ml of miR-214-enriched exosomes compared to the control exosomes (Fig. 4B). Therefore, this dose was used for vivo analysis. Mice received intraperitoneal injections of 100 μg of miR-214-enriched exosomes (n = 4), control exosomes (n = 4) or an equivalent volume of PBS (n = 4) every 2 days; these injections were initiated on Day 14 post implantation. Figure 4 View largeDownload slide Functional characterization of miR-214 enriched exosomes in endometriosis mice models. (A) The visible lesions within the peritoneal cavity of a mouse after 14 days of treatment. (B) Mice were treated with 0–100 μg/ml of miR-214 enriched exosomes. Expression of exosomal miR-214 were increased at 100 μg/ml (n = 3, *P < 0.001). (C) Immunostaining of Collagen αI and αSMA in endometrial-like lesions. (D) CTGF and Collagen αI expression were detected by RT-PCR analysis and normalized to GAPDH (n = 4, *P < 0.001 versus PBS group). (E) Collagen production were assessed by Sirius red staining, black outlined arrow indicates collagen, scale bar=50 μm. (F) Collagen production were assessed by Masson Trichrome stain, scale bar=50 μm. Figure 4 View largeDownload slide Functional characterization of miR-214 enriched exosomes in endometriosis mice models. (A) The visible lesions within the peritoneal cavity of a mouse after 14 days of treatment. (B) Mice were treated with 0–100 μg/ml of miR-214 enriched exosomes. Expression of exosomal miR-214 were increased at 100 μg/ml (n = 3, *P < 0.001). (C) Immunostaining of Collagen αI and αSMA in endometrial-like lesions. (D) CTGF and Collagen αI expression were detected by RT-PCR analysis and normalized to GAPDH (n = 4, *P < 0.001 versus PBS group). (E) Collagen production were assessed by Sirius red staining, black outlined arrow indicates collagen, scale bar=50 μm. (F) Collagen production were assessed by Masson Trichrome stain, scale bar=50 μm. Immunohistochemical staining showed the presence of CTGF and Collagen αI expression in the endometriotic lesions of nude mice. Immunohistochemistry revealed miR-214-enriched exosomes significantly down-regulated the expression of CTGF and Collagen αI, providing evidence that miR-214 treatment inhibits fibrosis in endometriosis in vivo (Fig. 4C). Statistics on fluorescent intensity from immunofluorescence images is shown in Fig. S1D. As assessed by RT-PCR, we showed that CTGF and Collagen αI mRNA levels were significantly diminished in mice treated with miR-214-enriched exosomes (Fig. 4D). Similarly, Sirius red and Masson trichrome staining also showed that levels of Collagen αI were lower in miR-214 enriched groups than in either control group (Fig. 4E and F). Discussion Endometriosis is often associated with other connective tissue disorders and progresses in a similar way to prominent fibrosis (Matsuzaki et al., 2010). This fibrous tissue may cause the main clinical problems associated with endometriosis such as pelvic pain and infertility (Giudice and Kao, 2004). Our data demonstrate that endometriosis fibrosis in 24 patients was associated with elevated expression of fibrotic markers Collagen αI, αSMA and CTGF. Recent reports have indicated that miRNAs are important regulators of gene expression during fibrogenesis (Braza-Boïls et al., 2014) but the underlying mechanisms have not been elucidated. In this study, we reported that endometriosis fibrosis was accompanied with diminished miR-214 expression. Combined with previous study of miR-214 conservatively targeting on CTGF 3′-UTR in liver fibrosis, our study provided a mechanism for the reciprocal pattern of expression between CTGF mRNA and miR-214 in endometriosis fibrosis. In our in vitro study, we showed that miR-214 impacts fibrogenic pathways in ESCs by direct effects on CTGF expression. In addition, we found that EECs could affect production of CTGF. Thus the role of miR-214 in EECs will require further study. Our findings revealed that exosomal delivery of miR-214 to ecto-ESCs resulted in decreased expression of CTGF mRNA, as well as of mRNAs for collagen. Exosomes are a class of nano-sized extracellular vesicles released from many different cell types providing for a novel mode of intercellular communication by containing molecules that regulate the functions of adjacent or remote cells (Wang et al., 2016). More recent studies have provided scientific evidence that exosomes contained cell-type specific collections of proteins, lipids and genetic material that could be transferred to target cells where they played a regulatory function (Chevillet et al., 2014). The range of biomolecules in exosomes is diverse, including soluble and membrane-bound proteins, lipids, mRNAs, microRNAs (miRNAs) and non-coding RNAs (ncRNAs). Signaling arises by surface-expressed ligands providing direct stimulation to the recipient cells or via the delivery of genetic information, receptors, functional proteins, or infectious agents into the recipient cells. It was an evolutionary adaptation for the signaling molecule to be well protected in the vesicle like exosomes whilst these biomolecules crossed the potential hostile extracellular environment (Lee et al., 2016). Here, we showed that miR-214 was expressed in Ecto-ESCs and could be transported in exosomes derived from them. Exosomal delivery of miR-214 to Ecto-ESCs resulted in decreased expression of endogenous CTGF mRNA, as well as the mRNA of collagen αI, both of which are fibrotic markers. In this study, the most significant finding was the identification of miR-214 as non-invasive gene therapy for endometrial fibrosis both in vitro and vivo and the evaluation of the effect of exosomal miR-214 on endometriosis fibrosis in an in vivo mouse model. Enhanced CTGF and Collagen αI production in euto-ESCs and Ecto-ESCs likely reflected diminishing endogenous levels of miR-214 in the cells themselves. Delivery of exosomal miR-214 to endometrial tissue may dampen the fibrogenic pathways thus limiting the magnitude of the fibrotic response. To determine if exosomal miR-214 could alter the extent of fibrosis in vivo, we used an Balb/c mouse endometriosis model to investigate disease-relevant fibrotic procession and determined the efficacy of the exosome therapeutic approach to treating endometriosis (Matsuzaki and Darcha, 2013). Progressive fibrosis and scarring was associated with the inflammation reaction and collagen deposition in the ectopic foci of endometriosis and surrounding tissues in the mouse model. Moreover, CTGF expression was also increased during fibrogenesis. In the group injected with miR-214 enriched exosomes, we observed that lower scores of fibrotic markers including CTGF and Collagen αI expression. MiR-214 enriched exosomes provided significant anti-fibrosis effect on in vivo model of endometriosis. In this paper, we confirmed the hypothesis that miR-214 was a potential therapeutic agent as an effective anti-fibrotic agent for endometriosiss. On the other hand, CTGF is also expressed in EECs, which provide a novel insight into paracrine signals of CTGF in endomeriosis fibrosis. The possibility of EECs as recipient cells for exosomes with miR-214 is worth developing further investigation. Recently, various innovative therapies involving exosome manipulation and subsequent reintroduction of exosome-based therapeutics into humans have been developed and validated (O'Loughlin et al., 2012). Exosomes have been identified in variety of body fluids, such us blood, urine, ascetic fluid, which indicates that the exchange of information between cells may occur via exosomes (Qazi et al., 2010). Emerging evidence supports the hypothesis that disease could be diagnosed by examining the levels of miRNAs in exosomes isolated from body fluids (Masyuk et al., 2013). Recent researches also revealed the potential utility of exosome-targeted therapies to control tissue injury and fibrosis (Borges et al., 2013). Although we have demonstrated that miR-214-enriched exosomes inhibited fibrogenesis in anendometriosis animal model, we intend to investigate the diagnosis effect and therapeutic application of exosomal miR-214 in women in future studies. Conclusively, exosomes-based therapeutic formulations have a potential to be a versatile strategy to treat fibrosis disorders of endometriosis. Supplementary data Supplementary data are available at Molecular Human Reproduction online. Acknowledgements We are very grateful to our gynecologcic colleagues for patient recruitment. Authors’ roles All authors were involved in designing and planning experiments, preparing and reviewing the article. D.W. carried out all the cultures, P.L. and X.M. analyzed data. All others approved the final article. Funding National Natural Science Foundation of China (Grant no. 81771549 Jinwei Miao). Conflict of interest None of the authors have any conflicts of interest to declare. References Abe W , Nasu K , Nakada C , Kawano Y , Moriyama M , Narahara H . MiR-196b targets c-myc and Bcl-2 expression, inhibits proliferation and induces apoptosis in endometriotic stromal cells . Hum Reprod 2013 ; 28 : 750 – 761 . Bartel DP . MicroRNAs: genomics, biogenesis, mechanism, and function . Cell 2004 ; 116 : 281 – 297 . Biernacka A , Dobaczewski M , Frangogiannis NG . TGF-β signaling in fibrosis . Growth Factors 2011 ; 29 : 196 – 202 . Borges FT , Melo SA , Ozdemir BC , Kato N , Revuelta I , Miller CA , Gattone VH 2nd , LeBleu VS , Kalluri R . TGF-β1-containing exosomes from injured epithelial cells activate fibroblasts to initiate tissue regenerative responses and fibrosis . J Am Soc Nephrol 2013 ; 24 : 385 – 392 . Braza-Boïls A , Marí-Alexandre J , Gilabert J , Sánchez-Izquierdo D , España F , Estellés A , Gilabert-Estellés J . MicroRNA expression profile in endometriosis: its relation to angiogenesis and fibrinolytic factors . Hum Reprod 2014 ; 29 : 978 – 988 . Chen L , Charrier A , Zhou Y , Chen R , Yu B , Agarwal K , Tsukamoto H , Lee LJ , Paulaitis ME , Brigstock DR . Epigenetic regulation of connective tissue growth factor by MicroRNA-214 delivery in exosomes from mouse or human hepatic stellate cells . Hepatology 2014 ; 59 : 1118 – 1129 . Chen L , Chen R , Kemper S , Charrier A , Brigstock DR . Suppression of fibrogenic signaling in hepatic stellate cells by Twist1-dependent microRNA-214 expression: role of exosomes in horizontal transfer of Twist1 . Am J Physiol Gastrointest Liver Physiol 2015 ; 309 : bG491 – bG499 . Chevillet JR , Kang Q , Ruf IK , Briggs HA , Vojtech LN , Hughes SM , Cheng HH , Arroyo JD , Meredith EK , Gallichotte EN et al. . Quantitative and stoichiometric analysis of the microRNA content of exosomes . Proc Natl Acad Sci USA 2014 ; 111 : 14888 – 14893 . Engels BM , Hutvagner G . Principles and effects of microRNA-mediated post-transcriptional gene regulation . Oncogene 2006 ; 25 : 6163 – 6169 . Giudice LC , Kao LC . Endometriosis . Lancet 2004 ; 364 : 1789 – 1799 . Harp D , Driss A , Mehrabi S , Chowdhury I , Xu W , Liu D , Garcia-Barrio M , Taylor RN , Gold B , Jefferson S et al. . Exosomes derived from endometriotic stromal cells have enhanced angiogenic effects in vitro . Cell Tissue Res 2016 ; 365 : 187 – 196 . Hsu CY , Hsieh TH , Tsai CF , Tsai HP , Chen HS , Chang Y , Chuang HY , Lee JN , Hsu YL , Tsai EM . MiRNA-199a-5p regulates VEGFA in endometrial mesenchymal stem cells and contributes to the pathogenesis of endometriosis . J Pathol 2014 ; 232 : 330 – 343 . Katsunari M , Tomoko M , Lukasz S , Kenneth E , Andrew L , Maria T . Anti-connective tissue growth factor (CTGF/CCN2) monoclonal antibody attenuates skin fibrosis in mice models of systemic sclerosis . Arthritis Res Ther 2017 ; 19 : 134 . Lee MJ , Park DH , Kang JH . Exosomes as the source of biomarkers of metabolic diseases . Ann Pediatr Endocrinol Metab 2016 ; 21 : 119 – 125 . Letelier P , Riquelme I , Hernández AH , Guzmán N , Farías JG , Roa JC . Circulating microRNAs as biomarkers in biliary tract cancers . Int J Mol Sci 2016 ; 17 : e791 . Lv JW , Wen W , Jiang C , Fu QB , Gu YJ , Lv TT , Li ZD , Xue W . Inhibition of microRNA-214 promotes epithelial-mesenchymal transition process and induces interstitial cystitis in postmenopausal women by upregulating Mfn2 . Exp Mol Med 2017 ; 49 : e357 . Malutan AM , Drugan T , Costin N , Ciortea R , Bucuri C , Rada MP , Mihu D . Pro-inflammatory cytokines for evaluation of inflammatory status in endometriosis . Cent Eur J Immunol 2015 ; 40 : 96 – 102 . Masyuk AI , Masyuk TV , Larusso NF . Exosomes in the pathogenesis, diagnostics and therapeutics of liver diseases . J Hepatol 2013 ; 59 : 621 – 625 . Matsuzaki S , Darcha C . Involvement of the Wnt/β-catenin signaling pathway in the cellular and molecular mechanisms offibrosis in endometriosis . PLoS One 2013 ; 8 : e76808 . Matsuzaki S , Darcha C . Anti-fibrotic properties of epigallocatechin-3-gallate in endometriosis . Hum Reprod 2014 ; 29 : 1677 – 1687 . Matsuzaki S , Darcha C , Maleysson E , Canis M , Mage G . Impaired down-regulation of E-cadherin and beta-catenin protein expression in endometrial epithelial cells in the mid-secretory endometrium of infertile patients with endometriosis . J Clin Endocrinol Metab 2010 ; 95 : 3437 – 3445 . Maybin JA , Barcroft J , Thiruchelvam U , Hirani N , Jabbour HN , Critchley HO . The presence and regulation of connective tissue growth factor in the human endometrium . Hum Reprod 2012 ; 27 : 1112 – 1121 . Mishra VV , Gaddagi RA , Aggarwal R , Choudhary S , Sharma U , Patel U . Prevalence; characteristics and management of endometriosis amongst infertile women: a one year retrospective study . J Clin Diagn Res 2015 ; 9 : QC01 – QC03 . O'Loughlin AJ , Woffindale CA , Wood MJ . Exosomes and the emerging field of exosome-based gene therapy . Curr Gene Ther 2012 ; 12 : 262 – 274 . Pillai RS . MicroRNA function: multiple mechanisms for a tiny RNA? RNA 2005 ; 11 : 1753 – 1761 . Qazi KR , Torregrosa Paredes P , Dahlberg B , Grunewald J , Eklund A , Gabrielsson S . Proinflammatory exosomes in bronchoalveolar lavage fluid of patients with sarcoidosis . Thorax 2010 ; 65 : 1016 – 1024 . Rebordão MR , Galvão A , Szóstek A , Amaral A , Mateus L , Skarzynski DJ , Ferreira-Dias G . Physiopathologic mechanisms involved in mare endometriosis . Reprod Domest Anim 2014 ; 49 : 82 – 87 . Sabuszewska-Jwiak A , Ciebiera M , Baran A , Jakiel G . Effectiveness of laparoscopic surgerie in treating infertility related to endometriosis . Ann Agric Environ Med 2015 ; 22 : 329 – 331 . Teague EM , Print CG , Hull ML . The role of microRNAs in endometriosis and associated reproductive conditions . Hum Reprod Update 2010 ; 16 : 142 – 165 . Wang Z , Chen JQ , Liu JL , Tian L . Exosomes in tumor microenvironment: novel transporters and biomarkers . J Transl Med 2016 ; 14 : 297 . Zhou Y , Zhou G , Tian C , Jiang W , Jin L , Zhang C , Chen X . Exosome-mediated small RNA delivery for gene therapy . Wiley Interdiscip Rev RNA 2016 ; 7 : 758 – 771 . © The Author(s) 2018. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: firstname.lastname@example.org This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)
Molecular Human Reproduction – Oxford University Press
Published: Apr 11, 2018
It’s your single place to instantly
discover and read the research
that matters to you.
Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.
All for just $49/month
Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly
Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.
Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.
Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.
All the latest content is available, no embargo periods.
“Hi guys, I cannot tell you how much I love this resource. Incredible. I really believe you've hit the nail on the head with this site in regards to solving the research-purchase issue.”Daniel C.
“Whoa! It’s like Spotify but for academic articles.”@Phil_Robichaud
“I must say, @deepdyve is a fabulous solution to the independent researcher's problem of #access to #information.”@deepthiw
“My last article couldn't be possible without the platform @deepdyve that makes journal papers cheaper.”@JoseServera