{"paper_id":"c125487b-5246-4a26-9c22-e846647b6793","body_text":"Endometriosis is a common and refractory estrogen-dependent gynecological disorder associated with dysmenorrhea, pelvic pain, and infertility 1 – 4 . Although benign, this chronic and multifactorial disease exhibits tumor-like biological behaviors and affects the physical and mental health of affected women 5 . However, the underlying pathogenesis and pathophysiology of endometriosis remain unclear.\nThe epithelial–mesenchymal transition (EMT) is a special biological process in which immotile epithelial cells convert into highly motile mesenchymal cells with migratory and invasive properties during embryonic development, chronic inflammation, tissue construction, fibrosis formation, and cancer metastasis 6 – 9 . The EMT is characterized by decreased expression of epithelial markers, such as E-cadherin, and increased expression of mesenchymal markers, such as N-cadherin and vimentin 10 . Many studies have documented the involvement of the EMT in the pathogenesis and development of endometriosis 7 – 9 , 11 . Chen et al. 12  first found decreased E-cadherin and increased vimentin expression in ectopic glandular epithelial cells in adenomyosis. Another study showed that E-cadherin-negative cells were frequently observed in endometriotic tissues, whereas N-cadherin, Twist, Slug, and Snail were all upregulated in endometriosis compared with that in the healthy endometrium 7 . The mesenchymal marker zinc finger E-box-binding homeobox 1 (ZEB1), a transcriptional repressor of E-cadherin, is also upregulated in endometriotic lesions 13 . Several signal pathways, including the Wnt/β-catenin pathway 11 , the Notch pathway 14 , and the transforming growth factor (TGF)-β/Smad pathway 15 , have been reported to regulate the EMT in endometriosis. Moreover, other factors, including estrogen 11 , 12  and hypoxia-inducible factor-1α 16 , may act as powerful inducers of the EMT in endometriosis. Changes in EMT marker proteins have been detected in ectopic epithelial cells, resulting in enhanced migration and invasion as a prerequisite for the establishment of endometriotic lesions.\nRecent studies have highlighted the regulatory mechanisms through which noncoding RNAs (ncRNAs) participate in the occurrence and progression of multiple diseases 17 – 19 . Circular RNAs (circRNAs), as a distinct type of ncRNAs, have a covalently closed continuous loop and display resistance to exonuclease digestion 20 , 21 . CircRNAs show highly stable expression in eukaryotic cells, are predominantly localized in the cytoplasm, and are evolutionarily conserved across species, suggestive of their important regulatory roles 17 . As competing endogenous RNAs (ceRNAs), circRNAs may sequester microRNAs (miRNAs) with their own miRNA response elements and function as miRNA “sponges” to strongly suppress miRNA activity, thereby increasing the levels of miRNA target genes and mediating cellular biological behaviors involved in human diseases 17 , 22 . For example,  ciRS-7  contains more than 70 selectively conserved miRNA target sites and regulates the initiation and progression of various malignancies in an  miR-7 -dependent manner 23 , 24 . In addition,  circ-PVT1  facilitates paclitaxel resistance by upregulating ZEB1 via  miR-124-3p  in gastric cancer 25 . Overexpression of  circ-0001649  inhibits the proliferation and migration of hepatocellular carcinoma (HCC) in vitro and in vivo by serving as a ceRNA to sponge  miR-127-5p ,  miR-612 , and  miR-4688 26 . These findings suggest that circRNAs may be potential biomarkers and therapeutic targets in the diagnosis and treatment of multiple diseases. However, our understanding of the relationships between circRNAs and endometriosis remains limited, and translating current circRNA-related research into clinical practice is challenging.\nOur previous study showed that circRNAs are aberrantly expressed in the ectopic endometrium (EcEM) compared with that in paired eutopic endometrium (EuEM) samples and demonstrated that  hsa-circ-0020093 , derived from the  ATRNL1  gene locus (thus, designated  circATRNL1 ), was upregulated in ovarian endometriosis 27 . Accordingly, in this study, we aimed to investigate the roles of  circATRNL1  in endometriosis in vitro. Our findings provided important insights into the roles of  circATRNL1  in the EMT in endometriosis and may aid in developing suitable therapeutic strategies.\n\nThis study was approved by the Ethics Committees of Shengjing Hospital of China Medical University (ethics no. 2018PS504K), and written informed consent was obtained from all patients before surgical procedures and sample collection.\nSnap-frozen cyst walls of ovarian endometriomas and matched EuEM samples from the same patient were collected from 60 women (20–44 years old) with a laparoscopic and histological diagnosis of stage III/IV endometriosis according to the Revised American Society for Reproductive Medicine classification system (rASRM; 1997). All patients had regular menstrual cycles (21–35 days), and none had received any hormone therapy for at least 6 months prior to the operation. All EuEM samples were collected during the proliferative phase of the menstrual cycle, as confirmed by both the date of the last menstrual period and histological diagnosis.\nIshikawa cells (a well-differentiated endometrial adenocarcinoma cell line) and HEK-293T cells were purchased from Huiying Biological Technology Co. Ltd (Shanghai, China) and cultured in modified Eagle’s medium (Gibco, USA) with 10% fetal bovine serum (FBS; Bioind, Israel), 100 μg/mL streptomycin, and 100 IU/mL penicillin (Beyotime, Shanghai, China) in a humidified atmosphere with 5% CO 2  at 37 °C.\nImmunohistochemical staining was performed on paraffin-embedded tissues. Three-micrometer-thick sections were cut and placed on glass slides. Tissues were deparaffinized in xylene and rehydrated in an ethanol gradient. Antigen retrieve was performed in 10 mM Sodium Citrate Buffer (pH 6.0). Overall, 3% H 2 O 2  was used to block endogenous peroxidase for 15 min. Nonspecific background staining was then blocked by incubation with goat serum (Solarbio, China) for 15 min. The slices were incubated with primary antibodies overnight at 4 °C and horseradish peroxidase (HRP)-conjugated secondary antibodies at 37 °C for 1 h. All primary and secondary antibodies were as follows: rabbit polyclonal anti-E-cadherin ( P12830 ) (Cat. No.WL01482, 1:200; Wanleibio, China), mouse monoclonal anti-N-cadherin ( P19022 ) (1:300; Proteintech, Wuhan, China), rabbit polyclonal anti-vimentin ( P08670 ) (1:200; Proteintech), mouse monoclonal anti-YAP1 ( P46937 ) (1:200; Proteintech), rabbit polyclonal anti-ZEB1 ( P37275 ) (1:200; Proteintech), goat anti-mouse IgG H&L antibody (Product # 31321, 1:500; Thermo Fisher, USA), and goat anti-rabbit IgG H&L antibody (Product # 31460, 1:500; Thermo Fisher, USA). All slides were incubated with DAB (Solarbio, China) and then counterstained with hematoxylin (Solarbio, China) before they were dehydrated and mounted. Finally, the staining was visualized under a light microscopy (Olympus, Japan). Three randomly selected microscopic fields (×400 magnification) were photographed and the mean optic density in each field was counted and analyzed using Image-Pro Plus software (Media Cybernetics, USA).\nThree interference sequences targeting  circATRNL1  were constructed and inserted into the  EcoR I– BamH I site of the pHBLV-U6-MCS-CMV-ZsGreen-PGK-Puro vector. The sequences were as follows: #1, 5′-ATAGTTGGCAAGGTTAACAGAACCT-3′; #2, 5′-TTGGCAAGGTTAACAGAACCTTCTG-3'; #3, 5'-CAATGATAGTTGGCAAGG TTAACAG-3′; For lentivirus-mediated overexpression of  circATRNL1  in Ishikawa cells, full-length  circATRNL1  was inserted into the pHBLV-CMV-circ-MCS-EF1-zsgreen-T2A-puro vector. Lentivirus packaging, cell infection, and selection of puromycin-resistant cells were performed according to the instruction manual provided by Hanbio Biotechnology (Shanghai, China). The sequences of miRNA mimics, miRNA inhibitors, and siRNAs were as follows:  miR-141-3p  mimic, forward 5′-UAACACUGUCUGGUAAAGAUGG-3′ and reverse 5′-AUCUUUACCAGACAGUGUUAUU-3′;  miR-141-3p  inhibitor, 5′-CCAUCUUUACCAGACAGUGUUA-3′;  miR-200a-3p  mimic, forward 5′-UAACACUGUCUGGUAAACG AUGU-3′ and reverse 5′-AUCGUUACCAGACAGUGUUAUU-3′;  miR-200a-3p  inhibitor, 5′-AC AUCGUUACCAGACAGUGUUA-3′; Yes-associated protein 1 ( YAP1 ) siRNA (#1), forward 5′-CUGCCACCAA GCUAGAUAATT-3′ and reverse 5′-UUAUCUAGCUUGGUGGCAGTT-3′; (#2), forward 5′-GGUGAUACUAUCAACCAAATT-3′ and reverse 5′-UUUGGUUGAUAGUAUCACCTT-3′; (#3), forward 5′-GACGACCAAUAGCUCAGAUTT-3′ and reverse 5′-AUCUGAGCUAUUGGUCGUCTT-3′; negative control (NC), forward 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse 5′-ACGUGACACGUUCGGAG AATT-3′; inhibitor negative control (si-NC), 5′-CAGUACUUUUGUGUAGUACAA-3′. All of these RNA oligos were purchased from GenePharma (Suzhou, China) and were transfected into Ishikawa cells using Lipofectamine 3000 Reagent (Invitrogen, USA).\nTotal RNA was isolated from human tissues and cultured cells with TRIzol reagent (TaKaRa, Japan). RNA quantity and quality were measured using a Nanophotometer N50 (Implen, Germany). Only when the ratio of the absorbance at 260 and 280 nm was between 1.8 and 2.2 was the total RNA sample considered acceptable. All RNA samples were stored at −80 °C until further use. The expression levels of circRNAs were evaluated by RT-qPCR using a One-step SYBR PrimeScript RT-PCR kit (cat. no. RR660A; TaKaRa) on an Applied Biosystems 7500 FAST instrument (Applied Biosystems, Foster City, CA, USA). The cycling program was initiated at 42 °C for 5 min and 95 °C for 10 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. An Mir-X miRNA First-strand Synthesis Kit (cat. no. 638315; TaKaRa) and PrimeScript RT Reagent Kit with gDNA Eraser (cat. no. RR047A; TaKaRa) were used for first-strand synthesis of miRNAs and mRNAs, respectively. Then, the amplification conditions were carried out using SYBR Premix Ex TaqII (cat. no. RR820A; TaKaRa) according to the manufacturer’s instructions, as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. All PCR primers were designed and synthesized by Sangon Biotech (Shanghai, China) and were as follows:  circATRNL1 , forward 5′-GCAATGATAGTTGCAAGGTTAAC-3′, reverse 5′-GCCTTCAATGAGC CAAGTACA-3′;  miR-141-3p , forward 5′-GCGCTAACACTGTCTGGTAAAGATGG-3′;  miR-200a-3p , forward 5′-GCGTAACACTGTCTGGTAACGATGT-3′;  YAP1 , forward 5′-CCGTTTCCCAGACTACCTT-3′, reverse 5′-TTGGCATCAGCTCCTCTC-3′. Each sample had three individual technical replicates. The threshold cycle method (2 −ΔΔCT ) was used to calculate relative quantification of expression levels compared with the internal standard.\nAfter transfection, Ishikawa cells were seeded on 96-well flat-bottomed microplates at a density of 5000 cells/well. The culture medium was regularly replaced. For analysis of cell proliferation, 100 μL of Cell Counting Kit-8 (CCK-8) reagent (Dojindo, Japan) was added into each well at different time points (0, 24, 48, 72, and 96 h), followed by incubation for 1 h at 37 °C. The absorbance of each well at 490 nm was observed and measured with a Universal Microplate Spectrophotometer (Bio-Tek Instruments, Inc., Winooski, VT, USA).\nCell invasion assays were performed using a Transwell system (Corning, NY, USA). After dilution with serum-free medium, 100 μL Matrigel (cat. no. 356234; Corning) was added to each transwell chamber and coagulated for 4 h at 37 °C. After treatment for 48 h, Ishikawa cells were washed and cultured with serum-free medium for 12 h. Next, a 200-μL aliquot of the above single-cell suspension (2 × 10 4  cells) was placed into each upper chamber of a transwell plate. The lower chamber was filled with 700 mL of medium containing 10% FBS. After 24 h of incubation, a cotton swab was used to remove cells remaining in the upper chamber. The cells that invaded into the membrane were fixed in 4% formaldehyde (Solarbio, Beijing) for 30 min, and then stained with 0.1% crystal violet solution (Solarbio). Eight randomly selected microscopic fields were photographed with an Eclipse Ti-s microscope (Nikon, Japan), and the number of cells in each field was counted using Image-Pro Plus software (Media Cybernetics, USA). Cell migration assays were performed according to the same procedure, except that the Matrigel was omitted.\nHybridization was performed overnight at 37 °C with specific  circATRNL1  probes, sections were washed, and DAPI staining was performed for 20 min in the dark. The sections were observed under a Leica TCS SP2 AOBS Confocal (upright) fluorescence microscope (×40 objective, ×10 ocular), and images were acquired. Nuclei stained with DAPI appeared blue under UV excitation and contained  circATRNL1 , which was labeled with Cy3 (red emission), and miRNAs ( miR-141-3p  and  miR-200a-3p ) which were labeled with FITC (green emission). The sequence of probes used for FISH were as follows: c ircATRNL1 , 5′-TTCTGTTAACCTTGCCAACTA-3′;  miR-141-3p , 5′-CCATCTTTACCAGACAGTGTTA-3′;  miR-200a-3p , 5′-CATCGTTACCAGACAGTGTTA-3′. All the probes synthesis and FISH detection were performed by Geneseed (Guangzhou, China).\npmiR-GLO-YAP1 wild-type or mutated vectors, miRNA mimics or negative control (Genepharm), and wild-type  circATRNL1  or mutated  circATRNL1  vectors (Ribobio, Guangzhou, China) were transfected into 293T cells. Firefly and Renilla luciferase activities were measured consecutively 24 h after transfection using Dual-Luciferase Reporter Assays (cat. no. E1910; Promega, Madison, WI, USA) according to the manufacturer’s instructions.\nTissues and cell lysates were prepared with RIPA buffer (Beyotime) containing 10% phenylmethylsulfonyl fluoride. Next, a BCA Kit (Beyotime) was used to quantify protein concentrations. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% gels (Beyotime) and transferred onto polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked with 5% skim milk for 2 h and incubated with primary antibodies at room temperature for 1.5 h or overnight at 4 °C. All primary and secondary antibodies were purchased from Proteintech (Wuhan, China). The antibodies were as follows: mouse monoclonal anti-E-cadherin (Cat.No. 60335-1-Ig, 1:2500), mouse monoclonal anti-N-cadherin (Cat.No. 66219-1-Ig,1:2000), mouse monoclonal anti-vimentin (Cat.No. 60330-1-Ig, 1:6000), mouse monoclonal anti-YAP1 (Cat.No. 66900-1-Ig,1:1000), rabbit polyclonal anti-ZEB1 (Cat.No.21544-1-AP, 1:1000), mouse monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ( P04406 ) (Cat.No.60004-1-Ig, 1:20000), mouse monoclonal anti-β-tubulin ( P04350 ) (Cat.No. 66240-1-Ig, 1:20000), goat anti-mouse IgG H&L (Cat.No. SA00001-1 HRP conjugated; 1:5000), and goat anti-rabbit IgG H&L antibodies (Cat.No. SA00001-2, HRP conjugated; 1:5000). The immunoblot signal was quantified with ImageJ software.\nAll experiments were performed in triplicate. The data are expressed as means ± standard errors of the means and analyzed by GraphPad Prism 7 (La Jolla, CA, USA). Differences between two groups were evaluated with Student’s  t  tests. Comparisons among three or more groups were performed by one-way analysis of variance and Tukey’s post hoc test. Each experiment was conducted triplicate. Correlations among  circATRNL1 ,  miR-141-3p/miR-200a-3p , and  YAP1  were analyzed with Spearman rank correlation.  P  values of less than 0.05 were considered statistically significant.\n\nTo validate the expression levels of  circATRNL1 , RT-qPCR was conducted in 60 EcEM and EuEM samples. The data showed that  circATRNL1  expression was significantly increased in ovary EcEM tissues compared with that in EuEM tissues (Fig.  1a ,  P  = 0.0002). This suggested that  circATRNL1  may play a role in the development of endometriosis. Fig. 1 circATRNL1 regulates proliferation, migration, invasion, and EMT process in endometriosis. a  The expression level of  circATRNL1  in 60 ovary endometriosis tissue samples was examined by using qRT-PCR.  b \n circATRNL1  was overexpressed in Ishikawa cells by transfecting with lentivirus-mediated vector pHBLV-ATRNL1. NC was used as a control.  c \n circATRNL1  was silenced in Ishikawa cells by transfecting with specific sh-ATRNL1. shRNA-NC was used as a control.  d  CCK-8 assays were conducted to determine the influence of  circATRNL1  on cell proliferation.  e  Transwell migration assays showed that  circATRNL1  regulated the migrative potential of Ishikawa cells. Photographs were taken at ×200 magnification. Scale bar represents 100 μm.  f  Transwell invasion assays showed that  circATRNL1  regulated the invasive potential of Ishikawa cells.  g  Western blot analysis was utilized to analyze the impacts of  circATRNL1  overexpression or knockdown on EMT progress in Ishikawa cells.  h  Immunohistochemistry of EMT markers in 60 EuEM and EcEM tissues. Original magnification: ×400. Scale bars represent 50 μm.  i  Western blot analysis of EMT markers in 60 EuEM and EcEM tissues. Assays were performed in triplicate. * P  < 0.05, ** P  < 0.01, *** P  < 0.001, **** P  < 0.0001 represent statistical difference.\na  The expression level of  circATRNL1  in 60 ovary endometriosis tissue samples was examined by using qRT-PCR.  b \n circATRNL1  was overexpressed in Ishikawa cells by transfecting with lentivirus-mediated vector pHBLV-ATRNL1. NC was used as a control.  c \n circATRNL1  was silenced in Ishikawa cells by transfecting with specific sh-ATRNL1. shRNA-NC was used as a control.  d  CCK-8 assays were conducted to determine the influence of  circATRNL1  on cell proliferation.  e  Transwell migration assays showed that  circATRNL1  regulated the migrative potential of Ishikawa cells. Photographs were taken at ×200 magnification. Scale bar represents 100 μm.  f  Transwell invasion assays showed that  circATRNL1  regulated the invasive potential of Ishikawa cells.  g  Western blot analysis was utilized to analyze the impacts of  circATRNL1  overexpression or knockdown on EMT progress in Ishikawa cells.  h  Immunohistochemistry of EMT markers in 60 EuEM and EcEM tissues. Original magnification: ×400. Scale bars represent 50 μm.  i  Western blot analysis of EMT markers in 60 EuEM and EcEM tissues. Assays were performed in triplicate. * P  < 0.05, ** P  < 0.01, *** P  < 0.001, **** P  < 0.0001 represent statistical difference.\nTo further explore the underlying functions of  circATRNL1  in endometriosis, we performed a series of validation assays by overexpressing and silencing  circATRNL1  in Ishikawa cells, respectively. The relative expression level of  circATRNL1  was validated by RT-qPCR. Importantly, the pHBLV-ATRNL1 vector upregulated  circATRNL1  in Ishikawa cells (Fig.  1b ,  P  = 0.0071). Three specific lentivirus-mediated shRNAs (sh1-ATRNL1, sh2-ATRNL1, and sh3-ATRNL1) were transfected into Ishikawa cells to silence  circATRNL1  expression; sh1 showed the highest efficiency and was selected for further cellular experiments (Fig.  1c ,  P  = 0.0009 for sh-NC vs. sh1,  P  = 0.0815 for sh-NC vs. sh2,  P  = 0.003 for sh-NC vs. sh3).\nSubsequently, CCK-8 assays were conducted to determine the influence of  circATRNL1  on cell proliferation. Our results demonstrated that overexpression of  circATRNL1  significantly promoted the proliferation of Ishikawa cells ( P  = 0.0010), whereas  circATRNL1  knockdown decreased the growth of Ishikawa cells (Fig.  1d ,  P  = 0.00005). We also observed that the migratory and invasive capabilities of Ishikawa cells were significantly enhanced by overexpression of  circATRNL2  (Fig.  1e ,  P  = 0.0108 for NC vs. pHBLV-ATRNL1,  P  = 0.0025 for sh-NC vs. sh1-ATRNL1), but significantly inhibited by silencing of  circATRNL1  (Fig.  1f ,  P  = 0.0025 for NC vs. pHBLV-ATRNL1,  P  = 0.0078 for sh-NC vs. sh1-ATRNL1). These results revealed that  circATRNL1  knockdown may efficiently reduce endometriosis by inhibiting cell growth, migration, and invasiveness.\nTo further detect whether  circATRNL1  promoted migration and invasion by facilitating the EMT in Ishikawa cells, we tested the expression of EMT hallmarks by western blot analysis. As shown in Fig.  1g , the levels of epithelial markers (E-cadherin) were enhanced, whereas the levels of mesenchymal markers (N-cadherin, vimentin, and ZEB1) were obviously decreased in  circATRNL1  knockdown Ishikawa cells. In contrast, the levels of EMT hallmarks showed the opposite changes in response to  circATRNL1  overexpression. Taken together, our findings demonstrated that  circATRNL1  expression was correlated with the EMT process in endometriosis. In addition, we tested the expression of EMT hallmarks by IHC and western blot analysis in 60 ovary EcEM tissues and EuEM tissues. The data showed that, compared with that in EuEM tissues, the levels of epithelial markers (E-cadherin) were decreased in EcEM tissues, whereas the levels of mesenchymal markers (N-cadherin, vimentin, and ZEB1) were obviously enhanced (Fig.  1h, i ).\nAccumulating evidence has demonstrated that circRNAs act as ceRNAs to sponge miRNAs and modulate the expression of target genes at the post-transcriptional level. To confirm that  circATRNL1  exerted biological functions through acting as a ceRNA, we first performed FISH assays to identify the localization of  circATRNL1  in Ishikawa cells.  CircATRNL1  was mainly localized in the cytoplasm of Ishikawa cells (Fig.  2a ). According to our previous bioinformatics prediction,  miR-141-3p  and  miR-200a-3p  may bind  circATRNL1  to affect the pathogenesis of endometriosis 27 . Furthermore, a luciferase reporter vector was designed and transfected into HEK-293T cells to facilitate the combination between  circATRNL1  and the two miRNAs (Fig.  2b ). The results showed that both  miR-141-3p  mimics ( P  = 0.0078) and  miR-200a-3p  mimics ( P  = 0.0008) decreased the luciferase activity of wild-type  circATRNL1  (circATRNL1-WT), whereas the luciferase activity of mutated  circATRNL1  (circATRNL1-MUT) was not significantly altered (Fig.  2c ). Subsequently,  miR-141-3p  ( P  < 0.00001) and  miR-200a-3p  ( P  < 0.00001) were shown to be downregulated in EcEM tissues compared with that in EuEM tissues (Fig.  2d ). Accordingly, the expression correlation between circATRNL1 and  miR-141-3p  ( r  = −0.434,  P  = 0.001) and  miR-200a-3p  ( r  = −0.418 , P  = 0.001) was separately proved to be negative. Fig. 2 miR-141-3p and miR-200a-3p are targets of circATRNL1. a  FISH assay was used to determine the location of  circATRNL1 ,  miR-141-3p , and  miR-200a-3p  in Ishikawa cells (blue, DAPI nuclear staining; red,  circATRNL1 ; green,  miR-141-3p  and  miR-200a-3p ). Scale bars represent 20 μm.  b  The binding sites between wild-type  circATRNL1  or mutated  circATRNL1  and  miR-141-3p/miR-200a-3p  were predicted by using bioinformatics analysis.  c  Dual-luciferase reporter assays were carried out in HEK-293T cell to confirm the combination between  circATRNL1  and  miR-141-3p  mimics or  miR-200a-3p  mimics.  d  qRT-PCR analysis was carried to determine the expression pattern of  miR-141-3p  and  miR-200a-3p  in ovary endometriosis tissues. **** P  < 0.0001 represents statistical difference.\na  FISH assay was used to determine the location of  circATRNL1 ,  miR-141-3p , and  miR-200a-3p  in Ishikawa cells (blue, DAPI nuclear staining; red,  circATRNL1 ; green,  miR-141-3p  and  miR-200a-3p ). Scale bars represent 20 μm.  b  The binding sites between wild-type  circATRNL1  or mutated  circATRNL1  and  miR-141-3p/miR-200a-3p  were predicted by using bioinformatics analysis.  c  Dual-luciferase reporter assays were carried out in HEK-293T cell to confirm the combination between  circATRNL1  and  miR-141-3p  mimics or  miR-200a-3p  mimics.  d  qRT-PCR analysis was carried to determine the expression pattern of  miR-141-3p  and  miR-200a-3p  in ovary endometriosis tissues. **** P  < 0.0001 represents statistical difference.\nmiR-141-3p  and  miR-200a-3p  belong to the  miR-200  family, which modulates EMT progression in several diseases. Nevertheless, the specific roles of  miR-141-3p  and  miR-200a-3p  in endometriosis are still unknown. Accordingly,  miR-141-3p  mimics/inhibitors and  miR-200a-3p  mimics/inhibitors were transfected into Ishikawa cells for overexpression or knockdown of the corresponding target gene, with mi-NC and in-NC used as the control group. RT-qPCR was used to measure transfection efficiency after 48 h. Co-transfection with pHBLV-ATRNL1 or sh1-ATRNL1 rescued the efficiency of miRNA mimics (Fig.  3a ,  P  = 0.0018 for  mi-NC  vs.  miR-141-3p  mimics,  P  = 0.0129 for  miR-141-3p  mimics vs. pHBLV-ATRNL1+  miR-141-3p  mimics,  P  = 0.0111 for  mi-NC  vs. pHBLV-ATRNL1+  miR-141-3p  mimics;  P  < 0.00001 for  mi-NC  vs.  miR-200a-3p  mimics,  P  < 0.00001 for  miR-200a-3p  mimics vs. pHBLV-ATRNL1+  miR-200a-3p  mimics,  P  = 0.0025 for  mi-NC  vs. pHBLV-ATRNL1+  miR-200a-3p  mimics) or inhibitors (Fig.  3b ,  P  = 0.002 for  in-NC  vs.  miR-141-3p  inhibitors,  P  = 0.0105 for  miR-141-3p  inhibitors vs. sh1-ATRNL1+  miR-141-3p  inhibitors,  P  = 0.0328 for  in-NC  vs. sh1-ATRNL1 +  miR-141-3p  inhibitors;  P  < 0.00001 for  in-NC  vs.  miR-200a-3p  inhibitors,  P  = 0.0002 for  miR-200a-3p  inhibitors vs. sh1-ATRNL1+  miR-200a-3p  inhibitors,  P  = 0.0001 for  in-NC  vs. sh1-ATRNL1+  miR-200a-3p  inhibitors). Similarly, CCK-8 assays and transwell migration and invasion assays were carried out to evaluate the influence of  miR-14-3p/miR-200a-3p  mimics on Ishikawa cells. As showed in Fig.  3c , cell proliferation was obviously inhibited by  miR-141-3p  mimics ( P  < 0.00001) and  miR-200a-3p  mimics ( P  < 0.00001). The results of transwell experiments demonstrated that the migratory (Fig.  3d ,  P  = 0.0001 for mi-NC vs.  miR-141-3p ,  P  = 0.0001 for mi-NC vs.  miR-200a-3p ) and invasive (Fig.  3e ,  P  = 0.0013 for mi-NC vs.  miR-141-3p ,  P  = 0.0072 for mi-NC vs.  miR-200a-3p ) capabilities of Ishikawa cells were dramatically suppressed in the context of  miR-141-3p  or  miR-200a-3p  overexpression. Finally, the results of western blot analysis demonstrated that EMT progression was inhibited in  miR-141-3p - or  miR-200a-3p -overexpressing Ishikawa cells (Fig.  3f ). Overall, these results suggested that both  miR-141-3p  and  miR-200a-3p  may exert inhibitory effects during the development of endometriosis. Fig. 3 miR-141-3p and miR-200a-3p inhibited EMT process in endometriosis. a \n miR-141-3p  and  miR-200a-3p  were overexpressed with  miR-141-3p  or  miR-200a-3p  mimics in Ishikawa cells, respectively, and be partly rescued by pHBLV-ATRNL1 co-transfection. mi-NC was taken as the control group.  b \n miR-141-3p  and  miR-200a-3p  were downregulated with  miR-141-3p  or  miR-200a-3p  inhibitors in Ishikawa cells, respectively, and be partly rescued by sh1-circATRNL1 co-transfection. in-NC was taken as the control group.  c – e  CCK-8 and transwell assays were separately carried out to identify the influence of  miR-141-3p  and  miR-200a-3p  mimics on cell proliferation, migration, and invasion. Photographs were taken at ×200 magnification. Scale bar represents 100 μm.  f  The effect of  miR-141-3p  and  miR-200a-3p  mimics on EMT progress in Ishikawa cells was evaluated through detecting the EMT marker proteins with western blot analysis. * P  < 0.05, ** P  < 0.01, *** P  < 0.001, **** P  < 0.0001 represent statistical difference.\na \n miR-141-3p  and  miR-200a-3p  were overexpressed with  miR-141-3p  or  miR-200a-3p  mimics in Ishikawa cells, respectively, and be partly rescued by pHBLV-ATRNL1 co-transfection. mi-NC was taken as the control group.  b \n miR-141-3p  and  miR-200a-3p  were downregulated with  miR-141-3p  or  miR-200a-3p  inhibitors in Ishikawa cells, respectively, and be partly rescued by sh1-circATRNL1 co-transfection. in-NC was taken as the control group.  c – e  CCK-8 and transwell assays were separately carried out to identify the influence of  miR-141-3p  and  miR-200a-3p  mimics on cell proliferation, migration, and invasion. Photographs were taken at ×200 magnification. Scale bar represents 100 μm.  f  The effect of  miR-141-3p  and  miR-200a-3p  mimics on EMT progress in Ishikawa cells was evaluated through detecting the EMT marker proteins with western blot analysis. * P  < 0.05, ** P  < 0.01, *** P  < 0.001, **** P  < 0.0001 represent statistical difference.\nBased on the ceRNA hypothesis, we used MiRWalk v3.0 ( http://mirwalk.umm.uni-heidelberg.de/ ), with strict screening conditions and three prediction algorithms (Targetscan, MiRDB, and mirTarbase), to predict downstream target mRNAs of  miR-141-3p  and  miR-200a-3p . Four mRNAs were found to be potential targets of  miR-141-3p , whereas three mRNAs were found to be potential targets of  miR-200a-3p  (Fig.  4a ). Among these seven mRNAs,  YAP1  was the only common target and was chosen for subsequent analyses. The binding sites for  miR-141-3p  and  miR-200a-3p  in the 3′-untranslated region (UTR) of  YAP1  were predicted (Fig.  4b ). Similarly, luciferase reporter assays were conducted to demonstrate the interactions between the two miRNAs and  YAP1 . The luciferase activity of wild-type  YAP1  (YAP1-WT) was significantly reduced in HEK-293T cells transfected with  miR-141-3p  mimics ( P  = 0.00001) or  miR-200a-3p  mimics ( P  = 0.00001), whereas that of mutated  YAP1  (YAP1-MUT) was not significantly altered (Fig.  4c ). Subsequently, high expression levels of YAP1 mRNA ( P  = 0.00004) and protein ( P  = 0.013) were detected in EcEM tissues compared with that in EuEM tissues (Fig.  4d–f ). Spearman correlation analysis proved negative relevance between YAP1 and  miR-141-3p  ( r  = −0.326,  P  = 0.012) as well as negative relevance between YAP1 and  miR-200a-3p  ( r  = −0.312,  P  = 0.015). On the contrary,  YAP1  expression correlation with  circATRNL1  was proved to be positive ( r  = 0.950,  P  < 0.0001). Fig. 4 YAP1 is a common target mRNA of miR-141-3p and miR-200a-3p. a  The downstream target mRNAs of  miR-141-3p/miR-200a-3p  were predicted by three bioinformatics tools (Targetscan, MiRDB, and mirTarbase).  b  The binding sites between 3′UTR of  YAP1  and  miR-141-3p/miR-200a-3p  were predicted by using bioinformatics analysis.  c  Dual-luciferase reporter assays were carried in HEK-293T cells which were co-transfected with  miR-141-3p  mimics or  miR-200a-3p  mimics and pmiR–pmiR-GLO-YAP1.  d  The expression levels of  YAP1  mRNA were tested in ovary endometriosis tissues through using qRT-PCR.  e ,  f  Immunohistochemistry and western blot analysis were used to test YAP1 protein in ovary endometriosis tissues. Immunohistochemistry photographs were taken at ×400 magnification. Scale bars represent 50 μm. **** P  < 0.0001 represents statistical difference.\na  The downstream target mRNAs of  miR-141-3p/miR-200a-3p  were predicted by three bioinformatics tools (Targetscan, MiRDB, and mirTarbase).  b  The binding sites between 3′UTR of  YAP1  and  miR-141-3p/miR-200a-3p  were predicted by using bioinformatics analysis.  c  Dual-luciferase reporter assays were carried in HEK-293T cells which were co-transfected with  miR-141-3p  mimics or  miR-200a-3p  mimics and pmiR–pmiR-GLO-YAP1.  d  The expression levels of  YAP1  mRNA were tested in ovary endometriosis tissues through using qRT-PCR.  e ,  f  Immunohistochemistry and western blot analysis were used to test YAP1 protein in ovary endometriosis tissues. Immunohistochemistry photographs were taken at ×400 magnification. Scale bars represent 50 μm. **** P  < 0.0001 represents statistical difference.\nThree siRNA sequences were constructed and transfected into Ishikawa cells to knockdown  YAP1  mRNA expression; YAP1-si#2 showed the highest inhibitory efficiency as demonstrated through RT-qPCR results (Fig.  5a ,  P  = 0.0034 for in-NC vs. YAP1-si#1,  P  = 0.00002 for in-NC vs. YAP1-si#2, and  P  = 0.0026 for in-NC vs. YAP1-si#3) and was selected for further cellular experiments. CCK-8 assays and transwell migration and invasion assays were carried out to evaluate the influence of  YAP1  siRNA on Ishikawa cells. As depicted in Fig.  5b , cell proliferation was obviously inhibited by YAP1-si#2 ( P  < 0.0001). The results of transwell experiments demonstrated that the migratory (Fig.  5c ,  P  = 0.0001) and invasive (Fig.  5d ,  P  = 0.0013) capabilities of Ishikawa cells were significantly inhibited in the context of  YAP1  knockdown. Finally, the results of western blot analysis demonstrated that EMT progression was inhibited in  YAP1 -silencing Ishikawa cells (Fig.  5e ). Overall, these results suggested that  YAP1  may promote EMT progression in endometriosis. Fig. 5 YAP1 silencing inhibited EMT process in endometriosis. a  Knockdown of  YAP1  expression with  YAP1  siRNA in Ishikawa cells. in-NC was taken as the control group.  b – d  CCK-8 and transwell assays were separately carried out to identify the influence of  YAP1 siRNA  on cell proliferation, migration, and invasion. Photographs were taken at ×200 magnification. Scale bar represents 100 μm.  e  The effect of  YAP1 siRNA  on EMT progress in Ishikawa cells was evaluated through detecting the EMT marker proteins with western blot analysis. ** P  < 0.01, **** P  < 0.0001 represent statistical difference.\na  Knockdown of  YAP1  expression with  YAP1  siRNA in Ishikawa cells. in-NC was taken as the control group.  b – d  CCK-8 and transwell assays were separately carried out to identify the influence of  YAP1 siRNA  on cell proliferation, migration, and invasion. Photographs were taken at ×200 magnification. Scale bar represents 100 μm.  e  The effect of  YAP1 siRNA  on EMT progress in Ishikawa cells was evaluated through detecting the EMT marker proteins with western blot analysis. ** P  < 0.01, **** P  < 0.0001 represent statistical difference.\nTo analyze the regulatory relationships among  circATRNL1 ,  miR-141-3p/miR-200a-3p , and  YAP1 , mRNA and protein levels of  YAP1  were measured, and rescue assays were performed in Ishikawa cells. As depicted in Fig.  6a, b , the increased mRNA and protein levels of  YAP1  caused by pHBLV-ATRNL1 was rescued by  miR-141-3p  or  miR-200a-3p  mimics or  YAP1 -si#2, and meanwhile,  miR-141-3p  or  miR-200a-3p  inhibitors could rescue effects of sh1-ATRNL1 on  YAP1 . According to the results of CCK-8 and transwell assays, the effects of pHBLV-ATRNL1 on promoting proliferation, migration, and invasion in Ishikawa cells could be partly rescued by  miR-141-3p  or  miR-200a-3p  mimics or YAP1-si#2. In addition,  miR-141-3p  or  miR-200a-3p  inhibitors could partly rescue the suppressive effects of silencing  circATRNL1  (Fig.  6c–e ). Similarly, western blot results showed that the enhancing effects of pHBLV-ATRNL1 on EMT progression were partly reversed by  miR-141-3p  or  miR-200a-3p  mimics or YAP1-si#2, whereas the inhibitory effects of sh1-ATRNL1 on EMT progression were partly reversed by  miR-141-3p  or  miR-200a-3p  inhibitors (Fig.  6f ). Based on these data, we confirmed that  circATRNL1  acted as a ceRNA to modulate endometriosis progression via the  miR-141-3p / miR-200a-3p / YAP1  axis (Fig.  6g ). Fig. 6 Function of circATRNL1–miR-141-3p/miR-200a-3p–YAP1 axis in endometriosis progression. a ,  b  mRNA level and protein level of  YAP1  were measured by using qRT-PCR and western blot in Ishikawa cells which were co-transfected with  miR-141-3p/miR-200a-3p  mimics and pHBLV-ATRNL1 or  miR-141-3p/miR-200a-3p  inhibitors and sh-ATRNL1 or  YAP1 -si and pHBLV-ATRNL1.  c – e  CCK-8 and transwell assays were separately performed to detect proliferation and motility ability of indicated Ishikawa cells. Photographs were taken at ×200 magnification. Scale bar represents 100 μm.  f  The effect of  circATRNL1–miR-141-3p/miR-200a-3p-YAP1  axis on EMT protein markers in Ishikawa cells was evaluated through western blot analysis.  g  The mechanism diagram was generated to illustrate the mechanism of  circATRNL1–miR-141-3p/miR-200a-3p–YAP1  axis in endometriosis. * P  < 0.05, ** P  < 0.01, *** P  < 0.001, **** P  < 0.0001 represent statistical difference.\na ,  b  mRNA level and protein level of  YAP1  were measured by using qRT-PCR and western blot in Ishikawa cells which were co-transfected with  miR-141-3p/miR-200a-3p  mimics and pHBLV-ATRNL1 or  miR-141-3p/miR-200a-3p  inhibitors and sh-ATRNL1 or  YAP1 -si and pHBLV-ATRNL1.  c – e  CCK-8 and transwell assays were separately performed to detect proliferation and motility ability of indicated Ishikawa cells. Photographs were taken at ×200 magnification. Scale bar represents 100 μm.  f  The effect of  circATRNL1–miR-141-3p/miR-200a-3p-YAP1  axis on EMT protein markers in Ishikawa cells was evaluated through western blot analysis.  g  The mechanism diagram was generated to illustrate the mechanism of  circATRNL1–miR-141-3p/miR-200a-3p–YAP1  axis in endometriosis. * P  < 0.05, ** P  < 0.01, *** P  < 0.001, **** P  < 0.0001 represent statistical difference.\n\nIn recent years, a growing body of evidence has indicated that circRNAs play crucial regulatory roles in the pathogenesis and development of various diseases, including both malignant and benign diseases 18 , 19 , 22 – 27 . The circRNA  circAF4  functions as an oncogene to regulate the expression of the MLL-AF4 fusion protein and inhibit MLL leukemia progression by sponging  miR-128-3p 28 . CircRNAs and miRNAs function together to serve as miRNA sponges through a ceRNA network. A recent study revealed that upregulation of  circRNA-100338  was associated with poor prognosis in patients with hepatitis B-related HCC by activating the mammalian target of rapamycin signaling pathway in HCC via the  circRNA-100338/miR-141-3p/RHEB  axis 29 . In a diabetic mouse myocardial fibrosis model, functional experimental results demonstrated that  circRNA-010567  silencing upregulated  miR-141  and downregulated TGF-β1 expression, thus suppressing myocardial fibrosis 30 .  Circ-ZEB1.33  is an oncogene that promotes HCC cell proliferation by enhancing the expression of cyclin-dependent kinases 6 through sponging  miR-200a-3p 31 . This inspired us to explore the underlying mechanisms and functions of circRNAs in endometriosis.\nIn this study, we found that compared with the EuEM in patients with endometriosis,  circATRNL1  was significantly elevated in the EcEM. According to the results of IHC and western blot assays, we found decreased E-cadherin and increased ZEB1, N-cadherin as well as vimentin expression in EcEM tissues compared with that in EuEM tissues, which was consistent with previous research results 7 , 11 – 13 . To further investigate the influence of  circATRNL1  dysregulation on endometrial epithelial cell activities, a series of functional assays were conducted in Ishikawa cells. The results demonstrated that knockdown of  circATRNL1  inhibited cell proliferation, migration, and invasion, whereas overexpression of  circATRNL1  promoted these cellular bioactivities. Concomitantly, knockdown of  circATRNL1  reduced the protein expression of N-cadherin, vimentin, and ZEB1, but increased the expression of E-cadherin. Therefore,  circATRNL1  may exhibit oncogene-like properties in endometriosis by regulating the EMT process.\nThrough FISH assays, we first showed that  circATRNL1  was mainly localized in the cytoplasm of Ishikawa cells, which further confirmed our hypothesis that  circATRNL1  may act as a ceRNA by binding with certain miRNAs. Next, bioinformatics analysis and luciferase reporter assays were utilized to identify the target miRNAs of  circATRNL1 . As a result,  miR - 141 - 3p  and  miR - 200a - 3p  were identified. Previous studies have revealed a large number of miRNAs showing expression level alterations between EuEM and EcEM tissues, indicating their involvement in the establishment and progression of endometriosis, such as angiogenesis, inflammation, and immune regulation 32 – 36 . For example, a recent cell type-specific analysis identified 149 abnormally expressed miRNAs in endometriotic lesions, including extensive upregulation of  miR-139-5p  and downregulation of  miR-375  compared with eutopic stromal cells, which are potentially involved in endometriosis-associated infertility or in the regulation of invasive growth and cell proliferation in endometriosis development 36 . In our study, we detected downregulation of both  miR - 141 - 3p  and  miR - 200a - 3p  in ectopic endometriotic tissues, consistent with previous research 34 – 36 . Furtherly, a negative expression correlation between circATRNL1 and  miR-141-3p  as well as circATRNL1 and  miR-200a-3p  in endometriosis tissues was separately proved.\nImportantly,  miR - 141 - 3p  and  miR - 200a - 3p  belong to the  miR-200  family, which has been reported to regulate the EMT and metastasis by targeting a cohort of target genes, such as ZEB1 and E-cadherin 8 , 16 , 37 . Moreover,  miR-200a-3p  and  miR-141-3p  are downregulated in the plasma samples of patients with endometriosis compared with that in healthy controls, suggesting that these miRNAs could be used as putative noninvasive diagnostic biomarkers of endometriosis 38 . Li et al. 39  found that inhibition of  miR-200a  could promote migration and high mobility group box 1 expression, while decreasing E-cadherin expression in HCC cell lines. Another study showed that EMT progression was inhibited in  miR-200a-3p -overexpressing papillary thyroid cancer (PTC) cells 40 . Similarly, in this study, we also observed the inhibitory effects of  miR-141-3p  and  miR-200a-3p  on the EMT in endometriosis, and the effects of  circATRNL1  knockdown on cell proliferation, invasion, and EMT suppression could be partially attenuated by  miR-141-3p/miR-200a-3p  inhibition.\nMiRNAs regulate gene expression post-transcriptionally by interacting with the 3′-UTRs of mRNAs and triggering either translational repression or mRNA degradation 36 . Through bioinformatics prediction,  YAP1  was found to be the common target of  miR-141-3p  and  miR-200a-3p .  YAP1  is a downstream oncogene of the Hippo pathway 41 . When activated,  YAP1  localizes to the nucleus and binds to transcription factors, such as TEA domain DNA-binding family of transcription factors (TEAD), and then drives tumor growth, metastasis, and senescence in cancer cell lines and induces the EMT in many types of tumors.  YAP1  induces the EMT in non-small cell lung cancer by regulating the transcription of Slug via interacting with TEAD 42 . In addition,  YAP1  is highly associated with HCC and PTC and frequently upregulated during tumor formation 40 , 43 . Song et al. 44  demonstrated that elevated expression of  YAP1  promotes endometrial stromal cell proliferation and blocks apoptosis in vitro and in vivo. In the current study, we found that  YAP1  mRNA and protein levels were positively regulated by  circATRNL1  in endometrial cell lines. The functional assay results furtherly demonstrated that knockdown of  YAP1  expression in Ishikawa cells could significantly inhibited cell proliferation, migration and invasion, and reversed the EMT process. Finally, rescue assays demonstrated that  circATRNL1  improved the EMT by modulating miR-141-3p/ miR-200a-3p / YAP1  in endometriosis.\nIn conclusion, our findings suggested that  circATRNL1  promoted the EMT in endometriosis by upregulating  YAP1  through sponging  miR-141-3p  and  miR-200a-3p . However, there were some limitations to this study. First, the sample size was relatively small for RT-qPCR, and only rASRM stage III/IV cases were included. Second, the tissue samples were only derived from ectopic cyst walls and EuEM and not from other types of endometriosis or healthy controls. Accordingly, further validation involving in larger cohorts of patients with different types of endometriosis and healthy controls is warranted to confirm the clinical value of  circATRNL1 . Moreover, we used Ishikawa cells rather than primary endometrial epithelial cells for functional experiments, which may lead to unreliable conclusions. Further detailed studies of the mechanisms and functions of  circATRNL1  are needed. With the deepening exploration of circRNAs, we expect to provide new ideas on pathogenesis and find new promising therapeutic targets for endometriosis.\n\nSupplementary figure 1 \n Supplementary figure 1 \n Supplementary figure 2 \n Supplementary figure 2\nSupplementary figure 1\nSupplementary figure 1\nSupplementary figure 2\nSupplementary figure 2","source_license":"CC-BY-4.0","license_restricted":false}