Adenosine deaminase family acting on RNA 1 may be a de novo target on endometriosis

Adenosine deaminase family acting on RNA 1 may be a de novo target on endometriosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Adenosine deaminase family acting on RNA 1 may be a de novo target on endometriosis Thuy Ha Vu, Keiichiro Nakamura, Kunitoshi Shigeyasu, Kotaro Kubo, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2861746/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Adenosine deaminase family acting on RNA 1 (ADAR1) expression was examined to determine its correlation with endometriosis. The biological functions and inhibitory effects of ADAR1 knockdown were investigated in the EMC100, EMC214, and EPC-1 cell lines. ADAR1 was examined in patients with and without endometriosis using reverse transcription polymerase chain reaction (RT-PCR). The apoptotic expression of ADAR1 small interfering RNA (siRNA) was confirmed using flow cytometry. The biological functions and inhibitory effects of ADAR1 knockdown were investigated using RT-PCR in immortalized human uterine endometrial cell lines. ADAR1 expression was significantly higher in patients with endometriosis than in those without (P = 0.044). The proportions of viable cells decreased to 60.8%, 61.9%, and 72.9% (mock) and 58.9%, 40.2% and 59.1% (control) of the control cell viability at 48 hours after transient transfection of the ADAR1 siRNA into EMC100, EMC214, and EPC-1 cell lines. ADAR1 knockdown led to apoptosis through MDA-5, RIG-I, PKR, IRF3, IRF7, Caspase3, Caspase7, and Caspase8 expression. ADAR1 could be a potential therapeutic target in endometriosis. ADAR1 Endometriosis potential therapeutic target Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Endometriosis is a chronic inflammatory disease associated with debilitating chronic pelvic pain, which affects 6–10% of women of reproductive age [ 1 ]. The main hypotheses regarding the etiopathogenesis of endometriosis include retrograde menstruation, coelomic metaplasia, and an embryonic origin. Mechanisms involved in this complex and multifactorial disease include estrogen dependence, an aberrant inflammatory response, abnormal angiogenesis, and genetic and epigenetic alterations [ 2 ]. Steroid hormones such as estrogen and progesterone affect numerous immune and inflammatory responses in endometriosis [ 3 ]. Steroids and their receptors affect endometriosis by regulating estrogen and progesterone levels, and “steroid-dependent” disorders are strongly associated with endometriosis [ 4 ]. Progesterone plays a crucial role in female reproduction via the progesterone receptor. Dienogest (17a-cyanomethyl-17b-hydroxy-estra-4,9-dien-3-one [DNG]) is a fourth-generation progestin with potent oral progestational activity [ 5 , 6 ]. DNG has been reported to be highly effective in the treatment of endometriosis [ 7 – 9 ]. However, several patients with endometriosis do not respond to progestin (P4) or DNG administration. Therefore, new therapeutic targets for endometriosis are required. The aim of this study was to discover new therapeutic targets for endometriosis, focusing on epigenetic factors. RNA editing is a recently identified epigenetic mechanism that regulates the posttranscriptional activity of essential genes by altering their amino acid sequences, leading to changes in gene expression [ 10 ]. One such RNA editing process, wherein the conversion of adenosine (A) to inosine (I) in primary RNA transcripts (A-to-I editing) is mediated by adenosine deaminase family acting on RNA (ADAR), leads to transcriptome diversification in human cells [ 11 ]. This family includes three enzymes: ADAR1, ADAR2, and ADAR3. ADAR1 and ADAR2 are ubiquitously expressed and exhibit catalytic activity [ 12 – 16 ]. ADAR1 suppresses interferon (IFN) expression and IFN-mediated activity and is the most abundant gene in humans [ 17 ]. There are almost no reports examined ADAR1 on endometriosis. Therefore, this study aimed to explore the role of ADAR1 in endometriosis. We further aimed to demonstrate the potential of ADAR1 as a new therapeutic target for endometriosis. Patients and methods Patients and tissue specimens The study was approved by the institutional ethics committee of Okayama University (approval number: K2212-042). Informed consent was obtained from all the participants. All procedures were performed following relevant ethical standards and institutional ethics committee regulations. Retroperitoneal adipose tissue from the peritoneum of the ovarian fossa (removed as part of surgical treatment) was collected from patients with or without endometriosis. Twenty female patients of reproductive age who underwent surgical treatment at the Okayama University Hospital between April 2014 and October 2022 were recruited into this study after providing informed consent. Subjects were classified into the endometriosis group (n = 15) and the control group (n = 5). Endometriotic lesions were diagnosed visually for endometriosis and confirmed via histopathology. RNA isolation and real-time quantitative PCR analyses Total RNA was isolated from the human samples and endometrial cell lines using the RNeasy Lipid Tissue Mini kit (QIAGEN, Hilden, Germany). The iTaq Universal SYBR Green OneStep Kit and the MiniOpticon Real-Time PCR System (Bio-Rad, CA, USA) were used for gene expression analysis by real-time quantitative PCR. GAPDH was used as a normalization control. Primer sequences for ADAR1, Interleukin (IL)-1β, IL-6, interdifferentiation-associated gene 5 (MDA5), Retinoic acid-inducible gene-I (RIG-I), Protein Kinase R (PKR), Interferon regulatory factor (IRF)3, IRF7, Caspase3, Caspase7, Caspase8, and GAPDH genes are shown in Supplementary Table 1. Reagents EMC100, EMC214, and EPC-1 cell lines were obtained from the Japanese Collection of Research Bioresources Cell Bank. RL95-2 endometrial cancer cell lines were obtained from American Type Culture Collection. The effect of estrogen, P4, and DRG was obtained from Fujifilm (Fujifilm, Osaka, Japan). Anti-ER alpha and anti-PR antibodies, GAPDH for Western blotting, were obtained from Cell Signaling Technology (Danvers, MA, USA). ADAR1 siRNA (siADAR1, sc-37657), or control siRNA, were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Cell culture The EMC100, EMC214, and EPC-1 immortalized human uterine endometrial progenitor cell line were maintained in Dulbecco’s modified eagle’s medium (DMEM)/F12 phenol red-free (Life Technologies, CA, USA), supplemented with 10% fetal bovine serum (FBS). Cell lines were maintained in a humidified incubator containing 5% CO2 at 37°C. Cells were used for functional experiments within three months of passaging, post-receipt. Each cell line were trypsinized and plated in culture dishes. Western blot analysis Proteins were assessed using western blotting analysis. Cells were extracted using a lysis buffer (RIPA Lysis and Extraction Buffer; Thermo Fisher Scientific, Waltham, MA, USA). Each protein was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Invitrolon PVDF, 0.45 µm; Invitrogen, Carlsbad, CA, USA). After being blocked in 5% skim milk in Tris-buffered saline containing 0.1% Tween‒ 20 for one h, the membranes were incubated overnight at four °C with the following primary antibodies: anti-ER alpha (1:1000), anti-PR (1:1000) and anti-GAPDH (1: 5000). Each membrane was washed and then incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) for one h. Signals were then detected using the Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA). The experiments were repeated at least three times. Small-interfering RNA (siRNA) transfection Each cell line was trypsinized and plated in culture dishes. At ~ 50% confluency, the cell lines were transfected with an annealed ADAR1 siRNA, control siRNA, or an empty vector (mock) for gene silencing (final concentration, 100 nmol/L), using a siRNA transfection reagent. MTS assay The effect of estrogen, P4, DRG, and ADAR1 siRNA on the cell proliferation of EMC100, EMC214, and EPC-1 cells was evaluated using the MTS assay (Promega, WI, USA). Each cell line was harvested and suspended in phenol red-free DMEM/F12 medium supplemented with 10% FBS and seeded into 96-well culture plates at a density of 5×10 3 cells/well. After the cells were cultured in phenol red-free DMEM/F12 medium supplemented with 10% FBS overnight, the cells were deprived of 10% FBS, incubated with the control and each concentration of estrogen, P4, and DRG, and then transiently transfected with the control siRNA and ADAR1 siRNA for 48 hours. After incubation with MTS for one hour, the absorbances were measured at a wavelength of 490 nm using an ELISA plate reader (Bio-Rad, CA, USA). Apoptosis assay The cell fluorescence was observed using a microscope (Olympus, Tokyo, Japan). Apoptosis and necroptosis were measured by staining with fluorescein isothiocyanate (FITC)-conjugated annexin V using a MEBCYTO Apoptosis kit (MBL International Corp., MA, USA). Furthermore, the apoptosis and necroptosis were analyzed with the FACS cytometer. Cell growth in monolayers For evaluation of cell growth in monolayers, cells were plated at a density of 2×104 cells/well in 6-well plates containing DMEM/F12 supplemented with 10% FBS. The cell numbers were counted in triplicate after 2, 4, and 6 days using a hemocytometer to assess cell proliferation. Statistical analysis Statistical analyses were performed using the Mann–Whitney U-test for comparisons with controls and one-factor ANOVA followed by Fisher's protected least significance difference test for all pairwise comparisons. The analyses were performed using StatView version 5.0 (Abacus Concepts, Berkeley, CA). Differences were considered significant at P < 0.05. RESULTS ADAR1 expression is positively correlated with endometriosis. ADAR1 expression was examined in endometriosis and non-endometriosis patients by RT-PCR. As expected, ADAR1 expression was significantly higher in endometriosis patients (P = 0.044) (Fig. 1 A). Furthermore, we also examined the relationship between ADAR1 expression and cytokines such as interleukin-1β and IL-6. We found that ADAR1 expression was significantly correlated to IL-1β (R = 0.600, R 2 = 0.360, P = 0.005) and IL-6 (R = 0.680, R 2 = 0.462, P = 0.001, Fig. 1 B). Estrogen strengthened ADAR1 and cytokines such as IL-1β and IL-6 expressions on immortalized human uterine endometrial cell lines. As shown in Fig. 2 A, EMC100, EMC214, EPC-1, and RL95-2 cells expressed estrogen receptor (ER)-alpha protein on western blot analysis. Moreover, estrogen at each concentration induced activation of ER in EMC100, EMC214, and EPC-1 cells, as shown in MTS assays (Fig. 2 B). Therefore, EMC100, EMC214, and EPC-1 cells expressed functional ER and were useful as a stable human epithelial cell system for the analysis of estrogen effects and functions. Furthermore, we investigated the changes in ADAR1 and cytokines such as IL-1β and IL-6 induced by estrogen. The efficiency of ADAR1, IL-1β, and IL-6 expression was confirmed by realtime PCR. Figure 2 C, ADAR1 expressions were significantly increased by 10 − 8 mol/l estrogen into EMC100, EMC214, and EPC-1 cell lines (P = 0.049, P = 0.012, and P = 0.047). IL-1β expressions were significantly increased by estrogen into the EPC-1cell line (P = 0.013), and EMC100 also tended to express IL-1β upon estrogen stimulation highly (P = 0.065). Moreover, IL-6 expressions were significantly increased by estrogen in EMC100 and EPC-1 cell lines as expected (P = 0.006, and P = 0.013, Fig. 2 C). P4 and DNG did not attenuate ADAR1 and cytokines such as IL-1B and IL-6 expressions in immortalized human uterine endometrial cell lines. As shown in Fig. 2 D, the RL95-2 cell line expressed PR protein in western blot analysis. On the other hand, EMC100, EMC214, and EPC-1 cell lines showed negative PR expression (Fig. 2 D). The effects of P4 and DNG on the cell proliferation were examined by MTS assay on EMC100, EMC214, and EPC-1 cell lines. None of the three cell lines showed effective suppression of 10 − 7 mol/l P4 or 10 − 7 mol/l DNG (Fig. 2 E). The effects of P4 and DNG on the expression of ADAR1, IL-1b, and IL-6 of EMC100, EMC214, and EPC-1 cells were examined by realtime PCR. ADAR1, IL-1b, and IL-6 expressions were not significantly decreased by 10 − 7 mol/l P4 or 10 − 7 mol/l DNG into EMC100, EMC214, and EPC-1 cell lines (Fig. 2 F). Therefore, in the three non-PR-expressing cell lines cell proliferation was not inhibited by P4 or DRG, nor did they act on ADAR1 or cytokines. Knockdown of ADAR1 attenuated ADAR1 expression on immortalized human uterine endometrial cell lines. We decided to perform ADAR1 knockdown in EMC100, EMC214, and EPC-1 cells to examine the expression changes. The knockdown efficiency of ADAR1 was confirmed by PCR. ADAR1 expression was significantly decreased by transfection of the ADAR1 siRNA into EMC100 and EMC214 (Mock: p = 0.015 and p = 0.046). Furthermore, ADAR1 expression also decreased by transfection of the ADAR1 siRNA into EPC-1 cell line (P = 0.088, Fig. 3 A). Knockdown of ADAR1 suppressed cell proliferation and increased apoptosis in immortalized human uterine endometrial cell lines. We determined the effects of ADAR1 on cell proliferation in immortalized human uterine endometrial cell lines. We performed an MTS assay after transient transfection of the ADAR1 siRNA into EMC100, EMC214, and EPC-1 cell lines. The number of viable cells decreased to 60.8, 61.9 and 72.9% (Mock), 58.9, 40.2 and 59.1% (Control) of that of the control cell viability at 48 hours after transient transfection of the ADAR1 siRNA into EMC100, EMC214, and EPC-1 cells, respectively (All mock and control; P < 0.001: Fig. 3 B). Therefore, the knockdown of ADAR1 suppressed the cell proliferation in immortalized human uterine endometrial cell lines. The following apoptosis profiles were obtained from transient transfection of the ADAR1 into EMC100, EMC214, and EPC-1 cells. ADAR1 siRNA increased the early and late apoptosis, compared with the mock and control of 41.07%, 25.47%, and 26.56% (Mock), 32.01%, 18.54%, and 24.17% (Control) for EMC100, EMC214, and EPC-1 cells. Therefore, ADAR1 siRNA was likely to regulate the early and late apoptosis in endometrial cell lines (Fig. 3 C). Knockdown of ADAR1 activated apoptosis-related pathway in immortalized human uterine endometrial cell lines. Based on these previous findings, we hypothesized that ADAR1 might suppress apoptosis in EMC100, EMC214, and EPC-1 cells by suppressing the dsRNA-sensing signaling pathway. ADAR1 was shown to suppress innate immunity primarily through the RIG-I-like receptor (RLR)-initiated cytosolic dsRNA-sensing signaling pathway, including MDA5, and RIG-I [ 18 , 19 ]. RIG-I, MDA-5, and PKR induce apoptosis [ 20 – 22 ]. To trace the steps in the apoptosis, we evaluated the activation of Caspase3, Caspase7, and Caspase8. We first examined the effects of ADAR1 knockdown on the expression of MDA5, RIG-I, PKR, IRF3, IRF7, Caspases3, Caspase7, and Caspase8 in EMC100, EMC214, and EPC-1 cell line. MDA5 expression significantly increased after transfection of ADAR1 siRNA into EMC100, EMC214 and EPC-1 cells (Mock; P < 0.001, P = 0.016, and P < 0.001, Control siRNA; P < 0.001, P = 0.061, and P < 0.001). RIG-I expression significantly increased after the transfection of ADAR1 siRNA into EPC-1 cells (Mock; P < 0.001, Control siRNA; P < 0.001). PKR expression significantly increased after transfection of ADAR1 siRNA into EMC100 and EPC-1 cells (Mock; P = 0.007, and P = 0.001, Control siRNA; P = 0.005 and P = 0.002). The expression of one of the IFN-stimulated genes, IRF3 and IRF7, which is downstream of MDA5, RIG-I, and PKR, also increased after transfection of the ADAR1 into EMC100 and EPC-1 cells (IRF3: Mock; P = 0.020 and P < 0.001, Control siRNA; P = 0.028 and P = 0.001; IRF7: Mock; P < 0.001 and P < 0.001, Control siRNA; P < 0.001 and P < 0.001). Moreover, IRF7 expression significantly increased after transfection of ADAR1 siRNA into EMC214 cells (Mock; P = 0.009, Control siRNA; P = 0.022). To trace the steps in the apoptosis, Caspase3, Caspase7, and Caspase8 expressions significantly increased after transfection of ADAR1 siRNA into EPC-1 cells (Mock; P < 0.001, P < 0.001, and P = 0.003, Control siRNA; P = 0.002, P < 0.001, and P = 0.006). Furthermore, Caspase3 expression significantly increased after transfection of ADAR1 siRNA into EMC100 and EMC214 cells (Mock; P < 0.001 and P = 0.002, Control siRNA; P < 0.001 and P = 0.005) (Fig. 4 A). These results suggest that suppression of ADAR1 activates the dsRNA-sensing signaling pathway, which in turn increases the expression of apoptosis factors of MDA5, RIG-I, PKR, Caspase 3, Caspase7 and Caspase8. The knockdown of ADAR1 inhibited cell growth. The effects of ADAR1 expression on monolayer growth were analyzed using transfection of ADAR1siRNA into EMC100, EMC214, and EPC-1 cell lines. Knockdown of ADAR1 significantly inhibited monolayer growth in EMC100, EMC214, and EPC-1 cells compared with mock and control siRNA (Day6 Mock: P < 0.001, P < 0.001, P = 0.008, siControl: P < 0.001, P < 0.001, P = 0.006, Fig. 4 B). DISCUSSION Steroids and their receptors are essential for the regulation of estrogen and progesterone levels in endometriosis, and steroid-dependent disorders are strongly associated with endometriosis [ 4 ]. Endometriosis is a steroid-dependent, chronic inflammatory disease that primarily affects women of reproductive age. Hormonal factors and inflammation are commonly involved in the regulation of endometriosis [ 23 ]. Capellino et al. reported that estrogen modulates macrophages to regulate immune responses via its functional receptors [ 24 ]. Thus, estrogen stimulation is beneficial for the establishment of endometriotic lesions. In contrast, progesterone plays a crucial role in female reproduction via the PR. P4 and DNG have been reported to be highly effective in the treatment of endometriosis [ 7 – 9 ]. However, in the absence of PR, mice did not respond to P4 or DNG administration. Therefore, new therapeutic targets for endometriosis are required. We focused on epigenetics and the aim of this study was to discover new therapeutic targets for endometriosis. RNA editing is a recently identified epigenetic mechanism that regulates the posttranscriptional activity of essential genes by altering their amino acid sequences, leading to changes in gene expression [ 10 ]. A-to-I RNA editing is catalyzed by adenosine deaminase ADAR enzymes that bind to and edit dsRNA. The A-to-I RNA editing of messenger RNA can alter its characteristics to promote a more aggressive phenotype. ADARs are also involved in immune recognition, which is primarily mediated by various IFN responses [ 25 , 26 ]. Chen et al. reported that high ADAR1 expression was significantly associated with endometriosis [ 27 ]. However, there are no reports on the mechanism of action of ADAR1 in endometriosis. Therefore, we investigated the biological function of ADAR1 in endometriosis. In the present study, ADAR1 expression was significantly higher in endometriotic specimens than in non-endometriotic specimens. ADAR1 expression was significantly correlated with IL-1β and IL-6 with endometriosis. Therefore, we examined whether ADAR1 was involved in steroid dependence in ER-positive and PR-negative immortalized human uterine endometrial gland-derived mesenchymal/progenitor cell lines. Estrogen acts on the ER and induces overexpression of ADAR1 and other cytokines. However, P4 and DNG were not functional in the PR-negative cell lines. Furthermore, ADAR1 expression was strongly associated with steroid dependence. ADAR1 is also involved in immune recognition, which is primarily explained by the IFN response in various cancer types [ 28 , 29 ]..ADAR1 activates the type-I IFN pathway via dsRNA sensors (MDA5, RIG-I, and PKR). RIG-I and MDA-5 were previously reported to promote pro-apoptotic signaling, known as type-I interferon–dependent apoptosis [ 30 ]. Similarly, activated PKR can induce apoptosis [ 17 , 31 ], indicating that the suppression of apoptosis could be a possible function of ADAR1 in endometriosis. Herein, ADAR1 knockdown increased MDA5, RIG-1, PKR, IRF3, IRF7, Caspase3, Caspase7, and Caspase8 expression in immortalized human uterine endometrial gland-derived mesenchymal/progenitor cell lines, leading to apoptosis. Interestingly, ADAR1 is expected to be a new therapeutic target for endometriosis that does not respond to P4 or DNG. The limitations of this study are: first, it is a single-center study, and second, it is a retrospective analysis. Large-scale prospective studies are required to ascertain further the role and clinical significance of ADAR1 in endometriosis. In summary, this study has revealed the critical role of ADAR1 in endometriosis. ADAR1 increases the potential of endometriosis through the inhibition of apoptosis and hence could be a potential therapeutic target in endometriosis. Conclusion To the best of our knowledge, the current study is one of the first to. investigated the biological function of ADAR1 in endometriosis. ADAR1 expression was significantly higher in endometriosis patients. ADAR1 expression was significantly correlated to IL-1β and IL-6. Knockdown of ADAR1 led to apoptosis through MDA-5, RIG-I, PKR, IRF3, IRF7, Caspase3, Caspase7, and Caspase8 expression into immortalized human uterine endometrial cell lines. ADAR1 could be a potential therapeutic target in endometriosis. Declarations Funding information This work was supported in part by Grants-in-Aid for Scientific Research (19K09753, 22K09619 to KN). Acknowledgments None. Conflicts of interest/Competing interests All authors (Vu Thuy Ha, Keiichiro Nakamura, Kunitoshi Shigeyasu, Kotaro Kubo, Chiaki Kashino, Hisashi Masuyama) declare that they currently retain position as an an officer, or student of Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences and they have financial relationship with these organizations unrelated to this article. Ethics Approval The current study was approved by the research ethical committee of Okayama University (approval number: K2212-042). 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A Z-DNA binding domain present in the human editing enzyme, double-stranded RNA adenosine deaminase. Proc Natl Acad Sci U S A. 1997:94(16);8421-8426. https://doi: 10.1073/pnas.94.16.8421. Herbert A. Mendelian disease caused by variants affecting recognition of Z-DNA and Z-RNA by the Zα domain of the double-stranded RNA editing enzyme ADAR.Eur J Hum Genet. 2020:28(1);114-117. https://doi:10.1038/s41431-019-0458-6. Desterro JM, Keegan LP, Lafarga M, Berciano MT, O'Connell M, Carmo-Fonseca M. Dynamic association of RNA-editing enzymes with the nucleolus. J. Cell Sci. 2003:116 (Pt 9);1805-1818. https://doi:10.1242/jcs.00371. Lambert AW, Pattabiraman DR, Weinberg RA. Emerging biological principles of metastasis. Cell. 2017:168(4);670-691. https://doi:10.1016/j.cell.2016.11.037. Supplementary Files supTable.1A.xlsx Supplementary Table 1. Primer sequences for PCR Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2861746","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":198808205,"identity":"92ad2a80-9914-495a-848d-5ae13d1c50ff","order_by":0,"name":"Thuy Ha Vu","email":"","orcid":"","institution":"Okayama University - Shikata Campus: Okayama Daigaku - Shikata Campus","correspondingAuthor":false,"prefix":"","firstName":"Thuy","middleName":"Ha","lastName":"Vu","suffix":""},{"id":198808206,"identity":"c174155a-2f94-4030-ba0d-4df2c61f1845","order_by":1,"name":"Keiichiro Nakamura","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-4609-5258","institution":"Okayama University - Shikata Campus: Okayama Daigaku - Shikata Campus","correspondingAuthor":true,"prefix":"","firstName":"Keiichiro","middleName":"","lastName":"Nakamura","suffix":""},{"id":198808207,"identity":"40e700fe-a7e2-4b5d-9023-56662f21edde","order_by":2,"name":"Kunitoshi Shigeyasu","email":"","orcid":"","institution":"Okayama University - Shikata Campus: Okayama Daigaku - Shikata Campus","correspondingAuthor":false,"prefix":"","firstName":"Kunitoshi","middleName":"","lastName":"Shigeyasu","suffix":""},{"id":198808208,"identity":"f153f1a0-c57b-487e-be17-3e9b1688d344","order_by":3,"name":"Kotaro Kubo","email":"","orcid":"","institution":"Okayama University - Shikata Campus: Okayama Daigaku - Shikata Campus","correspondingAuthor":false,"prefix":"","firstName":"Kotaro","middleName":"","lastName":"Kubo","suffix":""},{"id":198808209,"identity":"34669c8d-07ed-4acf-880e-cb42b8bdcd8b","order_by":4,"name":"Chiaki Kashino","email":"","orcid":"","institution":"Okayama University - Shikata Campus: Okayama Daigaku - Shikata Campus","correspondingAuthor":false,"prefix":"","firstName":"Chiaki","middleName":"","lastName":"Kashino","suffix":""},{"id":198808210,"identity":"d9f7f16f-1235-41cb-b9ba-b954a7612e05","order_by":5,"name":"Hisashi Masuyama","email":"","orcid":"","institution":"Okayama University - Shikata Campus: Okayama Daigaku - Shikata Campus","correspondingAuthor":false,"prefix":"","firstName":"Hisashi","middleName":"","lastName":"Masuyama","suffix":""}],"badges":[],"createdAt":"2023-04-26 03:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2861746/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2861746/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":36958818,"identity":"1afa374c-87c7-46d2-b767-0ed389497056","added_by":"auto","created_at":"2023-05-12 14:55:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":275645,"visible":true,"origin":"","legend":"\u003cp\u003eA. Tissue analysis of ADAR1 with 5 non-endometriosis and 15 endometriosis patients. B. Regression analysis for the ADAR1 and IL-1B into 5 non-endometriosis and 15 endometriosis patients. C. Regression analysis for the ADAR1 and IL-6 into 5 non-endometriosis and 15 endometriosis patients.\u003c/p\u003e","description":"","filename":"Fig1dpi300.png","url":"https://assets-eu.researchsquare.com/files/rs-2861746/v1/3b30aa0300e2428a323a4527.png"},{"id":36957894,"identity":"3f7419da-5d62-44ff-a2c7-aebb91247e4a","added_by":"auto","created_at":"2023-05-12 14:47:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":692350,"visible":true,"origin":"","legend":"\u003cp\u003eA. Western blot analysis of Estrogen Receptor-alpha expression on EMC100, EMC214, EPC-1 endometrial cell and RL95-2 endometrial cancer cell lines. B. \u003ca href=\"https://www.sciencedirect.com/topics/medicine-and-dentistry/mts-assay\"\u003eMTS\u003c/a\u003e\u003ca href=\"https://www.sciencedirect.com/topics/medicine-and-dentistry/mts-assay\"\u003e assays\u003c/a\u003e\u0026nbsp;of EMC100, EMC214 and EPC-1 endometrial cells by each concentrations estrogen (10\u003csup\u003e-5\u003c/sup\u003e-10\u003csup\u003e-10\u003c/sup\u003e mol/l) for 48\u0026nbsp;hours. C. \u003ca href=\"https://www.sciencedirect.com/topics/medicine-and-dentistry/western-blot\"\u003eReal\u003c/a\u003e\u003ca href=\"https://www.sciencedirect.com/topics/medicine-and-dentistry/western-blot\"\u003e time\u003c/a\u003e\u003ca href=\"https://www.sciencedirect.com/topics/medicine-and-dentistry/western-blot\"\u003e PCR\u003c/a\u003e\u003ca href=\"https://www.sciencedirect.com/topics/medicine-and-dentistry/western-blot\"\u003e analysis\u003c/a\u003e\u0026nbsp;of the ADAR1, IL-1b\u0026nbsp;and\u0026nbsp;IL-6 expression levels by 10\u003csup\u003e-8\u003c/sup\u003e mol/l E2 into EMC100, EMC214 and EPC-1 endometrial cells for 48\u0026nbsp;hours. D. Western blot analysis of Progesterone Receptor expression on EMC100, EMC214, EPC-1 endometrial cells and RL95-2 endometrial cancer cell lines. E. \u003ca href=\"https://www.sciencedirect.com/topics/medicine-and-dentistry/mts-assay\"\u003eMTS\u003c/a\u003e\u003ca href=\"https://www.sciencedirect.com/topics/medicine-and-dentistry/mts-assay\"\u003e assays\u003c/a\u003e\u0026nbsp;of EMC100, EMC214 and EPC-1 endometrial cells by 10\u003csup\u003e-7\u003c/sup\u003e mol/l P4 or 10\u003csup\u003e-7\u003c/sup\u003e mol/l DNG for 48\u0026nbsp;hours. F. \u003ca href=\"https://www.sciencedirect.com/topics/medicine-and-dentistry/western-blot\"\u003eReal\u003c/a\u003e\u003ca href=\"https://www.sciencedirect.com/topics/medicine-and-dentistry/western-blot\"\u003e time\u003c/a\u003e\u003ca href=\"https://www.sciencedirect.com/topics/medicine-and-dentistry/western-blot\"\u003e PCR\u003c/a\u003e\u003ca href=\"https://www.sciencedirect.com/topics/medicine-and-dentistry/western-blot\"\u003e analysis\u003c/a\u003e\u0026nbsp;of the ADAR1, IL-1b\u0026nbsp;and\u0026nbsp;IL-6 expression levels by 10\u003csup\u003e-7\u003c/sup\u003e mol/l P4 or 10\u003csup\u003e-7\u003c/sup\u003e mol/l DNG into EMC100, EMC214 and EPC-1 endometrial cells for 48\u0026nbsp;hours.\u003c/p\u003e","description":"","filename":"Fig2dpi300.png","url":"https://assets-eu.researchsquare.com/files/rs-2861746/v1/d3ef156124c7d7cafe16503d.png"},{"id":36957898,"identity":"b5115ffc-d861-4e2a-96d0-8c4af57202a8","added_by":"auto","created_at":"2023-05-12 14:47:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":556960,"visible":true,"origin":"","legend":"\u003cp\u003eA. \u0026nbsp;\u003ca href=\"https://www.sciencedirect.com/topics/medicine-and-dentistry/western-blot\"\u003eReal\u003c/a\u003e\u003ca href=\"https://www.sciencedirect.com/topics/medicine-and-dentistry/western-blot\"\u003etime\u003c/a\u003e\u003ca href=\"https://www.sciencedirect.com/topics/medicine-and-dentistry/western-blot\"\u003ePCR\u003c/a\u003e\u003ca href=\"https://www.sciencedirect.com/topics/medicine-and-dentistry/western-blot\"\u003eanalysis\u003c/a\u003e of the ADAR1 expression levels after transient transfection of the mock, control siRNA (siCon) or ADAR1 siRNA (siADAR1) into EMC100, EMC214 and EPC-1 endometrial cells for 48 hours. B. \u003ca href=\"https://www.sciencedirect.com/topics/medicine-and-dentistry/mts-assay\"\u003eMTS\u003c/a\u003e\u003ca href=\"https://www.sciencedirect.com/topics/medicine-and-dentistry/mts-assay\"\u003e assays\u003c/a\u003e of EMC100, EMC214 and EPC-1 endometrial cells after transient transfection of the mock, siCon or siADAR1 for 48 hours. C. Representative flow cytometric data for \u003ca href=\"https://www.sciencedirect.com/topics/medicine-and-dentistry/programmed-cell-death\"\u003eapoptosis\u003c/a\u003e of EMC100, EMC214 and EPC-1 endometrial cells after transient transfection of the mock, siCon and siADAR1 for 48 hours.\u003c/p\u003e","description":"","filename":"Fig3dpi300.png","url":"https://assets-eu.researchsquare.com/files/rs-2861746/v1/de18b5a6a6961ce2f6eecfa9.png"},{"id":36957895,"identity":"9e95cdce-bdf3-4a8b-8188-98a833cd2eac","added_by":"auto","created_at":"2023-05-12 14:47:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":739508,"visible":true,"origin":"","legend":"\u003cp\u003eA. Real-time PCR of EMC100, EMC214 and EPC-1 endometrial cells after transient transfection of the mock, control (siCon) and ADAR1 siRNA (siADAR1) for 48 hours. Real-time PCR of MDA5, RIG-I, PKR, IRF3, IRF7, Caspase3, Caspase7 and Caspase8 expression after transfection of the mock, iCon and siADAR1 intoEMC100, EMC214 and EPC-1 endometrial cells for 48hours. B. The cell growth in monolayers after transient transfection of the mock, siCon and siADAR1 into EMC100, EMC214 and EPC-1endometrial cells in DMEM/ham’s F12 mediumsupplemented with 10% FBS for 2, 4 and 6 days. Numbers represent the data from triplicate experiments.\u003c/p\u003e","description":"","filename":"Fig4dpi300.png","url":"https://assets-eu.researchsquare.com/files/rs-2861746/v1/9310fb44d4e5526f3f582c10.png"},{"id":39452994,"identity":"e38bf7b1-5d06-4582-8a6f-32337272b2e6","added_by":"auto","created_at":"2023-07-03 10:44:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1230429,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2861746/v1/c38ec0eb-31dd-4ee7-b6ed-ac73b6ed9fbc.pdf"},{"id":36958819,"identity":"e3b3eab9-f5b2-4f84-ba19-e443b9c3da25","added_by":"auto","created_at":"2023-05-12 14:55:01","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11391,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 1. Primer sequences for PCR\u003c/p\u003e","description":"","filename":"supTable.1A.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2861746/v1/a527057f7a2aa4cbd894e4ac.xlsx"}],"financialInterests":"","formattedTitle":"Adenosine deaminase family acting on RNA 1 may be a de novo target on endometriosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEndometriosis is a chronic inflammatory disease associated with debilitating chronic pelvic pain, which affects 6\u0026ndash;10% of women of reproductive age [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The main hypotheses regarding the etiopathogenesis of endometriosis include retrograde menstruation, coelomic metaplasia, and an embryonic origin. Mechanisms involved in this complex and multifactorial disease include estrogen dependence, an aberrant inflammatory response, abnormal angiogenesis, and genetic and epigenetic alterations [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSteroid hormones such as estrogen and progesterone affect numerous immune and inflammatory responses in endometriosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Steroids and their receptors affect endometriosis by regulating estrogen and progesterone levels, and \u0026ldquo;steroid-dependent\u0026rdquo; disorders are strongly associated with endometriosis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Progesterone plays a crucial role in female reproduction via the progesterone receptor. Dienogest (17a-cyanomethyl-17b-hydroxy-estra-4,9-dien-3-one [DNG]) is a fourth-generation progestin with potent oral progestational activity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. DNG has been reported to be highly effective in the treatment of endometriosis [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, several patients with endometriosis do not respond to progestin (P4) or DNG administration. Therefore, new therapeutic targets for endometriosis are required. The aim of this study was to discover new therapeutic targets for endometriosis, focusing on epigenetic factors. RNA editing is a recently identified epigenetic mechanism that regulates the posttranscriptional activity of essential genes by altering their amino acid sequences, leading to changes in gene expression [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. One such RNA editing process, wherein the conversion of adenosine (A) to inosine (I) in primary RNA transcripts (A-to-I editing) is mediated by adenosine deaminase family acting on RNA (ADAR), leads to transcriptome diversification in human cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This family includes three enzymes: ADAR1, ADAR2, and ADAR3. ADAR1 and ADAR2 are ubiquitously expressed and exhibit catalytic activity [\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. ADAR1 suppresses interferon (IFN) expression and IFN-mediated activity and is the most abundant gene in humans [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. There are almost no reports examined ADAR1 on endometriosis. Therefore, this study aimed to explore the role of ADAR1 in endometriosis. We further aimed to demonstrate the potential of ADAR1 as a new therapeutic target for endometriosis.\u003c/p\u003e"},{"header":"Patients and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients and tissue specimens\u003c/h2\u003e \u003cp\u003e The study was approved by the institutional ethics committee of Okayama University (approval number: K2212-042). Informed consent was obtained from all the participants. All procedures were performed following relevant ethical standards and institutional ethics committee regulations. Retroperitoneal adipose tissue from the peritoneum of the ovarian fossa (removed as part of surgical treatment) was collected from patients with or without endometriosis. Twenty female patients of reproductive age who underwent surgical treatment at the Okayama University Hospital between April 2014 and October 2022 were recruited into this study after providing informed consent. Subjects were classified into the endometriosis group (n\u0026thinsp;=\u0026thinsp;15) and the control group (n\u0026thinsp;=\u0026thinsp;5). Endometriotic lesions were diagnosed visually for endometriosis and confirmed via histopathology.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRNA isolation and real-time quantitative PCR analyses\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTotal RNA was isolated from the human samples and endometrial cell lines using the RNeasy Lipid Tissue Mini kit (QIAGEN, Hilden, Germany). The iTaq Universal SYBR Green OneStep Kit and the MiniOpticon Real-Time PCR System (Bio-Rad, CA, USA) were used for gene expression analysis by real-time quantitative PCR. GAPDH was used as a normalization control. Primer sequences for ADAR1, Interleukin (IL)-1β, IL-6, interdifferentiation-associated gene 5 (MDA5), Retinoic acid-inducible gene-I (RIG-I), Protein Kinase R (PKR), Interferon regulatory factor (IRF)3, IRF7, Caspase3, Caspase7, Caspase8, and GAPDH genes are shown in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eReagents\u003c/h3\u003e\n\u003cp\u003eEMC100, EMC214, and EPC-1 cell lines were obtained from the Japanese Collection of Research Bioresources Cell Bank. RL95-2 endometrial cancer cell lines were obtained from American Type Culture Collection. The effect of estrogen, P4, and DRG was obtained from Fujifilm (Fujifilm, Osaka, Japan). Anti-ER alpha and anti-PR antibodies, GAPDH for Western blotting, were obtained from Cell Signaling Technology (Danvers, MA, USA). ADAR1 siRNA (siADAR1, sc-37657), or control siRNA, were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eThe EMC100, EMC214, and EPC-1 immortalized human uterine endometrial progenitor cell line were maintained in Dulbecco\u0026rsquo;s modified eagle\u0026rsquo;s medium (DMEM)/F12 phenol red-free (Life Technologies, CA, USA), supplemented with 10% fetal bovine serum (FBS). Cell lines were maintained in a humidified incubator containing 5% CO2 at 37\u0026deg;C. Cells were used for functional experiments within three months of passaging, post-receipt. Each cell line were trypsinized and plated in culture dishes.\u003c/p\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cp\u003eProteins were assessed using western blotting analysis. Cells were extracted using a lysis buffer (RIPA Lysis and Extraction Buffer; Thermo Fisher Scientific, Waltham, MA, USA). Each protein was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Invitrolon PVDF, 0.45 \u0026micro;m; Invitrogen, Carlsbad, CA, USA). After being blocked in 5% skim milk in Tris-buffered saline containing 0.1% Tween‒ 20 for one h, the membranes were incubated overnight at four \u0026deg;C with the following primary antibodies: anti-ER alpha (1:1000), anti-PR (1:1000) and anti-GAPDH (1: 5000). Each membrane was washed and then incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) for one h. Signals were then detected using the Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA). The experiments were repeated at least three times.\u003c/p\u003e\n\u003ch3\u003eSmall-interfering RNA (siRNA) transfection\u003c/h3\u003e\n\u003cp\u003eEach cell line was trypsinized and plated in culture dishes. At ~\u0026thinsp;50% confluency, the cell lines were transfected with an annealed ADAR1 siRNA, control siRNA, or an empty vector (mock) for gene silencing (final concentration, 100 nmol/L), using a siRNA transfection reagent.\u003c/p\u003e\n\u003ch3\u003eMTS assay\u003c/h3\u003e\n\u003cp\u003eThe effect of estrogen, P4, DRG, and ADAR1 siRNA on the cell proliferation of EMC100, EMC214, and EPC-1 cells was evaluated using the MTS assay (Promega, WI, USA). Each cell line was harvested and suspended in phenol red-free DMEM/F12 medium supplemented with 10% FBS and seeded into 96-well culture plates at a density of 5\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well. After the cells were cultured in phenol red-free DMEM/F12 medium supplemented with 10% FBS overnight, the cells were deprived of 10% FBS, incubated with the control and each concentration of estrogen, P4, and DRG, and then transiently transfected with the control siRNA and ADAR1 siRNA for 48 hours. After incubation with MTS for one hour, the absorbances were measured at a wavelength of 490 nm using an ELISA plate reader (Bio-Rad, CA, USA).\u003c/p\u003e\n\u003ch3\u003eApoptosis assay\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe cell fluorescence was observed using a microscope (Olympus, Tokyo, Japan). Apoptosis and necroptosis were measured by staining with fluorescein isothiocyanate (FITC)-conjugated annexin V using a MEBCYTO Apoptosis kit (MBL International Corp., MA, USA). Furthermore, the apoptosis and necroptosis were analyzed with the FACS cytometer.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eCell growth in monolayers\u003c/h3\u003e\n\u003cp\u003eFor evaluation of cell growth in monolayers, cells were plated at a density of 2\u0026times;104 cells/well in 6-well plates containing DMEM/F12 supplemented with 10% FBS. The cell numbers were counted in triplicate after 2, 4, and 6 days using a hemocytometer to assess cell proliferation.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using the Mann\u0026ndash;Whitney U-test for comparisons with controls and one-factor ANOVA followed by Fisher's protected least significance difference test for all pairwise comparisons. The analyses were performed using StatView version 5.0 (Abacus Concepts, Berkeley, CA). Differences were considered significant at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cb\u003eADAR1 expression is positively correlated with endometriosis.\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eADAR1 expression was examined in endometriosis and non-endometriosis patients by RT-PCR. As expected, ADAR1 expression was significantly higher in endometriosis patients (P\u0026thinsp;=\u0026thinsp;0.044) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Furthermore, we also examined the relationship between ADAR1 expression and cytokines such as interleukin-1β and IL-6. We found that ADAR1 expression was significantly correlated to IL-1β (R\u0026thinsp;=\u0026thinsp;0.600, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.360, P\u0026thinsp;=\u0026thinsp;0.005) and IL-6 (R\u0026thinsp;=\u0026thinsp;0.680, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.462, P\u0026thinsp;=\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cb\u003eEstrogen strengthened ADAR1 and cytokines such as IL-1β and IL-6 expressions on immortalized human uterine endometrial cell lines.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, EMC100, EMC214, EPC-1, and RL95-2 cells expressed estrogen receptor (ER)-alpha protein on western blot analysis. Moreover, estrogen at each concentration induced activation of ER in EMC100, EMC214, and EPC-1 cells, as shown in MTS assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Therefore, EMC100, EMC214, and EPC-1 cells expressed functional ER and were useful as a stable human epithelial cell system for the analysis of estrogen effects and functions. Furthermore, we investigated the changes in ADAR1 and cytokines such as IL-1β and IL-6 induced by estrogen. The efficiency of ADAR1, IL-1β, and IL-6 expression was confirmed by realtime PCR. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, ADAR1 expressions were significantly increased by 10\u0026thinsp;\u0026minus;\u0026thinsp;8 mol/l estrogen into EMC100, EMC214, and EPC-1 cell lines (P\u0026thinsp;=\u0026thinsp;0.049, P\u0026thinsp;=\u0026thinsp;0.012, and P\u0026thinsp;=\u0026thinsp;0.047). IL-1β expressions were significantly increased by estrogen into the EPC-1cell line (P\u0026thinsp;=\u0026thinsp;0.013), and EMC100 also tended to express IL-1β upon estrogen stimulation highly (P\u0026thinsp;=\u0026thinsp;0.065). Moreover, IL-6 expressions were significantly increased by estrogen in EMC100 and EPC-1 cell lines as expected (P\u0026thinsp;=\u0026thinsp;0.006, and P\u0026thinsp;=\u0026thinsp;0.013, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eP4 and DNG did not attenuate ADAR1 and cytokines such as IL-1B and IL-6 expressions in immortalized human uterine endometrial cell lines.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, the RL95-2 cell line expressed PR protein in western blot analysis. On the other hand, EMC100, EMC214, and EPC-1 cell lines showed negative PR expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The effects of P4 and DNG on the cell proliferation were examined by MTS assay on EMC100, EMC214, and EPC-1 cell lines. None of the three cell lines showed effective suppression of 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e mol/l P4 or 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e mol/l DNG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The effects of P4 and DNG on the expression of ADAR1, IL-1b, and IL-6 of EMC100, EMC214, and EPC-1 cells were examined by realtime PCR. ADAR1, IL-1b, and IL-6 expressions were not significantly decreased by 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e mol/l P4 or 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e mol/l DNG into EMC100, EMC214, and EPC-1 cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Therefore, in the three non-PR-expressing cell lines cell proliferation was not inhibited by P4 or DRG, nor did they act on ADAR1 or cytokines.\u003c/p\u003e \u003cp\u003e \u003cb\u003eKnockdown of ADAR1 attenuated ADAR1 expression on immortalized human uterine endometrial cell lines.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe decided to perform ADAR1 knockdown in EMC100, EMC214, and EPC-1 cells to examine the expression changes. The knockdown efficiency of ADAR1 was confirmed by PCR. ADAR1 expression was significantly decreased by transfection of the ADAR1 siRNA into EMC100 and EMC214 (Mock: p\u0026thinsp;=\u0026thinsp;0.015 and p\u0026thinsp;=\u0026thinsp;0.046). Furthermore, ADAR1 expression also decreased by transfection of the ADAR1 siRNA into EPC-1 cell line (P\u0026thinsp;=\u0026thinsp;0.088, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eKnockdown of ADAR1 suppressed cell proliferation and increased apoptosis in immortalized human uterine endometrial cell lines.\u003c/b\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWe determined the effects of ADAR1 on cell proliferation in immortalized human uterine endometrial cell lines. We performed an MTS assay after transient transfection of the ADAR1 siRNA into EMC100, EMC214, and EPC-1 cell lines. The number of viable cells decreased to 60.8, 61.9 and 72.9% (Mock), 58.9, 40.2 and 59.1% (Control) of that of the control cell viability at 48 hours after transient transfection of the ADAR1 siRNA into EMC100, EMC214, and EPC-1 cells, respectively (All mock and control; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001: Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Therefore, the knockdown of ADAR1 suppressed the cell proliferation in immortalized human uterine endometrial cell lines.\u003c/p\u003e \u003cp\u003eThe following apoptosis profiles were obtained from transient transfection of the ADAR1 into EMC100, EMC214, and EPC-1 cells. ADAR1 siRNA increased the early and late apoptosis, compared with the mock and control of 41.07%, 25.47%, and 26.56% (Mock), 32.01%, 18.54%, and 24.17% (Control) for EMC100, EMC214, and EPC-1 cells. Therefore, ADAR1 siRNA was likely to regulate the early and late apoptosis in endometrial cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003cb\u003eKnockdown of ADAR1 activated apoptosis-related pathway in immortalized human uterine endometrial cell lines.\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eBased on these previous findings, we hypothesized that ADAR1 might suppress apoptosis in EMC100, EMC214, and EPC-1 cells by suppressing the dsRNA-sensing signaling pathway. ADAR1 was shown to suppress innate immunity primarily through the RIG-I-like receptor (RLR)-initiated cytosolic dsRNA-sensing signaling pathway, including MDA5, and RIG-I [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. RIG-I, MDA-5, and PKR induce apoptosis [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. To trace the steps in the apoptosis, we evaluated the activation of Caspase3, Caspase7, and Caspase8. We first examined the effects of ADAR1 knockdown on the expression of MDA5, RIG-I, PKR, IRF3, IRF7, Caspases3, Caspase7, and Caspase8 in EMC100, EMC214, and EPC-1 cell line. MDA5 expression significantly increased after transfection of ADAR1 siRNA into EMC100, EMC214 and EPC-1 cells (Mock; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, P\u0026thinsp;=\u0026thinsp;0.016, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Control siRNA; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, P\u0026thinsp;=\u0026thinsp;0.061, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). RIG-I expression significantly increased after the transfection of ADAR1 siRNA into EPC-1 cells (Mock; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Control siRNA; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). PKR expression significantly increased after transfection of ADAR1 siRNA into EMC100 and EPC-1 cells (Mock; P\u0026thinsp;=\u0026thinsp;0.007, and P\u0026thinsp;=\u0026thinsp;0.001, Control siRNA; P\u0026thinsp;=\u0026thinsp;0.005 and P\u0026thinsp;=\u0026thinsp;0.002). The expression of one of the IFN-stimulated genes, IRF3 and IRF7, which is downstream of MDA5, RIG-I, and PKR, also increased after transfection of the ADAR1 into EMC100 and EPC-1 cells (IRF3: Mock; P\u0026thinsp;=\u0026thinsp;0.020 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Control siRNA; P\u0026thinsp;=\u0026thinsp;0.028 and P\u0026thinsp;=\u0026thinsp;0.001; IRF7: Mock; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Control siRNA; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Moreover, IRF7 expression significantly increased after transfection of ADAR1 siRNA into EMC214 cells (Mock; P\u0026thinsp;=\u0026thinsp;0.009, Control siRNA; P\u0026thinsp;=\u0026thinsp;0.022). To trace the steps in the apoptosis, Caspase3, Caspase7, and Caspase8 expressions significantly increased after transfection of ADAR1 siRNA into EPC-1 cells (Mock; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and P\u0026thinsp;=\u0026thinsp;0.003, Control siRNA; P\u0026thinsp;=\u0026thinsp;0.002, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and P\u0026thinsp;=\u0026thinsp;0.006). Furthermore, Caspase3 expression significantly increased after transfection of ADAR1 siRNA into EMC100 and EMC214 cells (Mock; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and P\u0026thinsp;=\u0026thinsp;0.002, Control siRNA; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and P\u0026thinsp;=\u0026thinsp;0.005) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). These results suggest that suppression of ADAR1 activates the dsRNA-sensing signaling pathway, which in turn increases the expression of apoptosis factors of MDA5, RIG-I, PKR, Caspase 3, Caspase7 and Caspase8.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cb\u003eThe knockdown of ADAR1 inhibited cell growth.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe effects of ADAR1 expression on monolayer growth were analyzed using transfection of ADAR1siRNA into EMC100, EMC214, and EPC-1 cell lines. Knockdown of ADAR1 significantly inhibited monolayer growth in EMC100, EMC214, and EPC-1 cells compared with mock and control siRNA (Day6 Mock: P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, P\u0026thinsp;=\u0026thinsp;0.008, siControl: P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, P\u0026thinsp;=\u0026thinsp;0.006, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eSteroids and their receptors are essential for the regulation of estrogen and progesterone levels in endometriosis, and steroid-dependent disorders are strongly associated with endometriosis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Endometriosis is a steroid-dependent, chronic inflammatory disease that primarily affects women of reproductive age. Hormonal factors and inflammation are commonly involved in the regulation of endometriosis [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Capellino et al. reported that estrogen modulates macrophages to regulate immune responses via its functional receptors [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Thus, estrogen stimulation is beneficial for the establishment of endometriotic lesions. In contrast, progesterone plays a crucial role in female reproduction via the PR. P4 and DNG have been reported to be highly effective in the treatment of endometriosis [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, in the absence of PR, mice did not respond to P4 or DNG administration. Therefore, new therapeutic targets for endometriosis are required. We focused on epigenetics and the aim of this study was to discover new therapeutic targets for endometriosis. RNA editing is a recently identified epigenetic mechanism that regulates the posttranscriptional activity of essential genes by altering their amino acid sequences, leading to changes in gene expression [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. A-to-I RNA editing is catalyzed by adenosine deaminase ADAR enzymes that bind to and edit dsRNA. The A-to-I RNA editing of messenger RNA can alter its characteristics to promote a more aggressive phenotype. ADARs are also involved in immune recognition, which is primarily mediated by various IFN responses [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Chen et al. reported that high ADAR1 expression was significantly associated with endometriosis [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, there are no reports on the mechanism of action of ADAR1 in endometriosis. Therefore, we investigated the biological function of ADAR1 in endometriosis.\u003c/p\u003e \u003cp\u003eIn the present study, ADAR1 expression was significantly higher in endometriotic specimens than in non-endometriotic specimens. ADAR1 expression was significantly correlated with IL-1β and IL-6 with endometriosis. Therefore, we examined whether ADAR1 was involved in steroid dependence in ER-positive and PR-negative immortalized human uterine endometrial gland-derived mesenchymal/progenitor cell lines. Estrogen acts on the ER and induces overexpression of ADAR1 and other cytokines. However, P4 and DNG were not functional in the PR-negative cell lines. Furthermore, ADAR1 expression was strongly associated with steroid dependence.\u003c/p\u003e \u003cp\u003eADAR1 is also involved in immune recognition, which is primarily explained by the IFN response in various cancer types [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]..ADAR1 activates the type-I IFN pathway via dsRNA sensors (MDA5, RIG-I, and PKR). RIG-I and MDA-5 were previously reported to promote pro-apoptotic signaling, known as type-I interferon\u0026ndash;dependent apoptosis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Similarly, activated PKR can induce apoptosis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], indicating that the suppression of apoptosis could be a possible function of ADAR1 in endometriosis. Herein, ADAR1 knockdown increased MDA5, RIG-1, PKR, IRF3, IRF7, Caspase3, Caspase7, and Caspase8 expression in immortalized human uterine endometrial gland-derived mesenchymal/progenitor cell lines, leading to apoptosis. Interestingly, ADAR1 is expected to be a new therapeutic target for endometriosis that does not respond to P4 or DNG.\u003c/p\u003e \u003cp\u003eThe limitations of this study are: first, it is a single-center study, and second, it is a retrospective analysis. Large-scale prospective studies are required to ascertain further the role and clinical significance of ADAR1 in endometriosis.\u003c/p\u003e \u003cp\u003eIn summary, this study has revealed the critical role of ADAR1 in endometriosis. ADAR1 increases the potential of endometriosis through the inhibition of apoptosis and hence could be a potential therapeutic target in endometriosis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTo the best of our knowledge, the current study is one of the first to. investigated the biological function of ADAR1 in endometriosis. ADAR1 expression was significantly higher in endometriosis patients. ADAR1 expression was significantly correlated to IL-1β and IL-6. Knockdown of ADAR1 led to apoptosis through MDA-5, RIG-I, PKR, IRF3, IRF7, Caspase3, Caspase7, and Caspase8 expression into immortalized human uterine endometrial cell lines. ADAR1 could be a potential therapeutic target in endometriosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported in part by Grants-in-Aid for Scientific Research (19K09753, 22K09619 to KN).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan lang=\"\"\u003eAcknowledgments\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest/Competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors (Vu Thuy Ha, Keiichiro Nakamura, Kunitoshi Shigeyasu, Kotaro Kubo, Chiaki Kashino, Hisashi Masuyama) declare that they currently retain position as an an officer, or student of Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences and they have financial relationship with these organizations unrelated to this article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEthics Approval\u0026nbsp;\u003c/strong\u003eThe current study was approved by the research ethical committee of Okayama University\u0026nbsp;(approval number: K2212-042).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConsent to Participate\u003c/strong\u003e All participants signed an informed consent.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConsent for Publication\u003c/strong\u003e All authors agree to transfer the copyright of the current study to the journal upon acceptance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAvailability of data and material \u0026nbsp;\u003c/strong\u003eAll authors can show the data of the study upon reasonable request from the editorial board of the journal.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCode Availability\u003c/strong\u003e SPSS version 25 (Statistical package for social science) for windows 10).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZondervan KT, Becker CM, Missmer SA. Endometriosis. N. Engl. J. Med. 2020;382(13):1244-1256. https://doi:10.1056/NEJMra1810764.\u003c/li\u003e\n\u003cli\u003eReis FM, Petraglia F, Taylor RN. Endometriosis: hormone regulation and clinical consequences of chemotaxis and apoptosis. Hum Reprod Update. 2013;19(4):406-18. https://doi:10.1093/humupd/dmt010.\u003c/li\u003e\n\u003cli\u003eStraub RH. The complex role of estrogen in inflammation. Endocr Rev. 2007;28(5):521-574. https://doi:10.1210/er.2007-0001.\u003c/li\u003e\n\u003cli\u003eYilmaz BD, Bulun SE. Endometriosis and nuclear receptors. Hum. Reprod. Update 2019;25(4):473-485. https://doi:10.1093/humupd/dmz005.\u003c/li\u003e\n\u003cli\u003eSitruk-Ware R. New progestogens: a review of their effects in perimenopausal and postmenopausal women. Drugs Aging 2004; 21(13):865-883. https://doi: 10.2165/00002512-200421130-00004.\u003c/li\u003e\n\u003cli\u003eSasagawa S, Shimizu Y, Kami H, Takeuchi T, Mita S, Imada K, et al. 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Cell. 2017:168(4);670-691. https://doi:10.1016/j.cell.2016.11.037.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ADAR1, Endometriosis, potential therapeutic target","lastPublishedDoi":"10.21203/rs.3.rs-2861746/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2861746/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAdenosine deaminase family acting on RNA 1 (ADAR1) expression was examined to determine its correlation with endometriosis. The biological functions and inhibitory effects of ADAR1 knockdown were investigated in the EMC100, EMC214, and EPC-1 cell lines. ADAR1 was examined in patients with and without endometriosis using reverse transcription polymerase chain reaction (RT-PCR). The apoptotic expression of ADAR1 small interfering RNA (siRNA) was confirmed using flow cytometry. The biological functions and inhibitory effects of ADAR1 knockdown were investigated using RT-PCR in immortalized human uterine endometrial cell lines. ADAR1 expression was significantly higher in patients with endometriosis than in those without (P\u0026thinsp;=\u0026thinsp;0.044). The proportions of viable cells decreased to 60.8%, 61.9%, and 72.9% (mock) and 58.9%, 40.2% and 59.1% (control) of the control cell viability at 48 hours after transient transfection of the ADAR1 siRNA into EMC100, EMC214, and EPC-1 cell lines. ADAR1 knockdown led to apoptosis through MDA-5, RIG-I, PKR, IRF3, IRF7, Caspase3, Caspase7, and Caspase8 expression. ADAR1 could be a potential therapeutic target in endometriosis.\u003c/p\u003e","manuscriptTitle":"Adenosine deaminase family acting on RNA 1 may be a de novo target on endometriosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-12 14:46:56","doi":"10.21203/rs.3.rs-2861746/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"02bcc065-6f55-4e36-941d-f2bcd48b93a5","owner":[],"postedDate":"May 12th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-07-03T10:44:08+00:00","versionOfRecord":[],"versionCreatedAt":"2023-05-12 14:46:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2861746","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2861746","identity":"rs-2861746","version":["v1"]},"buildId":"B-jG_2CBjPDmsCi4Wdhf-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}