Hsa_circ_0079474 facilitates epithelial-mesenchymal transition in intrauterine adhesion via miR-630/YAP1 axis

Hsa_circ_0079474 facilitates epithelial-mesenchymal transition in intrauterine adhesion via miR-630/YAP1 axis | 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 Article Hsa_circ_0079474 facilitates epithelial-mesenchymal transition in intrauterine adhesion via miR-630/YAP1 axis Chen Xing, Yan Zhou, Jiwen Wang, Zhenzhen Song, Jing Yang, Wei Xu, and 18 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3767908/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Insufficient understanding exists of the molecular mechanisms underlying circRNA involvement in IUA and requires further investigation. This research aims to examine the role of hsa_circ_0079474 (circDGKB-009) and its potential mechanisms in intrauterine adhesion (IUA). A circRNA microarray was utilized to identify differences in circRNA expression between fibrotic endometrial samples and normal endometrial samples. Subsequent studies confirmed the expression and biological functions of hsa_circ_0079474 both in vivo and in vitro using various experimental techniques such as CCK-8, EdU, flow cytometry, FISH, RT-PCR, Western blot and IHC/ICC. The interactions between hsa_circ_0079474 and miR-630, as well as miR-630 and YAP1 were determined using dual-luciferase reporter assay and RNA immunoprecipitation. Hsa_circ_0079474 was dramatically elevated in IUA tissues compared to normal tissues. Hsa_circ_0079474 was found to enhance cell proliferation, expedite cell cycle progression, and facilitate epithelial-mesenchymal transition (EMT). Mechanistically, hsa_circ_0079474 acted as a sponge for miR-630, resulting in upregulation of YAP1 expression. This, in turn, promoted the progression of IUA. Hsa_circ_0079474 improves IUA by regulating the miR-630/YAP1 axis, providing a novel understanding of the molecular mechanisms underlying circRNA in IUA. Health sciences/Diseases/Reproductive disorders Health sciences/Biomarkers/Diagnostic markers Intrauterine adhesion fibrosis hsa_circ_0079474 miR-630 YAP1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Intrauterine adhesion (IUA) is characterized by the development of endometrial fibrosis in uterine cavity and cervix which is derived from damage to the basal layer of the endometrium. With the rapid growth of artificial abortion, curettage and hysteroscopic operations, the incidence of IUA is explosive growing. Approximately 40% of females with incomplete abortions who experienced curettage developed IUA [ 1 – 3 ]. Since most cases are asymptomatic, predicting the true prevalence of IUA is difficult. A variety of treatments including hysteroscopy, intrauterine devices and hormonal therapies have been applied in ameliorating IUA, but showed limited effects for women with moderate and severe IUA especially, which also had high recurrence rate [ 4 ]. Therefore, it is essential to better understand the mechanisms of IUA and explore more alternative therapies to improve the outcome of menstruation and pregnancy. Epithelial-mesenchymal transition (EMT) is a biological phenomenon involving epithelial cells losing their typical epithelial phenotype and acquiring mesenchymal characteristics such as proliferative, migratory and invasive properties. EMT is involved in various pathophysiological process including embryogenesis, tissue construction, formation of fibrosis and cancer metastasis. EMT is identified by reduced levels of epithelial cell markers like E-cadherin and cytokeratin, accompanied by elevated levels of mesenchymal cell markers such as alpha smooth muscle actin (α-SMA) and Vimentin (VIM) [ 5 , 6 ]. EMT had also been observed playing an important role in the pathogenesis of endometrial fibrosis during IUA [ 7 , 8 ]. However, EMT remains to be studied further in order to determine the exact mechanisms involved in IUA development. Circular RNAs (circRNAs) are a subclass of non-coding RNAs which are characterized by forming a covalently closed loop structure, resulting resistance to exonuclease digestion [ 9 ]. Recently, circRNAs have been implicated in multiple pathophysiological processes in numerous studies. However, only circPlekha7 [ 10 ], circPTP4A2 [ 11 ] and circPTPN12 [ 12 ] were reported to be involved in IUA. There is still a lack of understanding regarding the molecular mechanisms underlying circRNAs in the process of IUA. MicroRNAs (miRNAs) are another type of non-coding RNAs which consist of 20–23 nucleotides. MiRNAs take participate in various biological process via negative regulation of target genes by binding to complementary sequences within mRNA targets at the level of post-transcription [ 13 ]. CircRNAs have miRNA response elements and may play regulatory role by sponging miRNAs to neutralize miRNA-mediated effects on downstream mRNAs, which is the main regulatory mechanisms of circRNAs in the numerous physiological and pathological processes [ 14 , 15 ]. In the current research, we examined a screening of circRNA expression patterns in IUA tissues and normal endometrium using a circRNA microarray. Following filtration and verification, we confirmed that hsa_circ_0079474, a circRNA originated from the exons 6–13 of human gene DGKB (diacylglycerol kinase beta), which was also known as circDGKB-009, exhibited significantly higher expression levels in IUA tissues compared to normal endometrium. Subsequently, we delved into the mechanism by which hsa_circ_0079474 contributes to the development of IUA and discovered that it exerts fibrotic effects by acting as a miR-630 sponge to activate its target gene yes-associated protein-1 (YAP1). These findings provided novel insights into circRNA-mediated IUA regulation and exploring new promising biomarkers for diagnosis of IUA. Results Hsa_circ_0079474 is upregulated in IUA patients Since EMT is a pivotal event in the progression of fibrosis, the EMT hallmarks including epithelial cell markers (E-cadherin and CK-18) and mesenchymal cell markers (α-SMA and VIM) were determined using qRT-PCR (Fig. 1A), Western blot (Fig. 1B) and immunohistochemistry (IHC) (Fig. 1C) techniques in both IUA tissues and normal tissues. The results, as depicted in Fig. 1a-1c, demonstrated a reduction of the levels of E-cadherin and CK-18 whereas the levels of α-SMA and VIM were obviously enhanced in IUA tissues compared to normal tissues, suggesting involvement of EMT in IUA progression. To identify the circRNAs expression profiles involved in IUA progression, a circRNA microarray analysis on 3 paired IUA samples and adjacent normal samples under hysteroscopy was performed. The criteria for selecting differentially expressed circRNAs was absolute fold change > 2.0 and p value less than 0.05. Among them, 348 differentially expressed circRNAs were reported in circBase (www.circbase.org/) and 235 differentially expressed circRNAs were detected in all samples including 72 upregulated circRNAs and 163 downregulated circRNAs. A heatmap was constructed to show different expressed circRNAs (Fig. 1D). Furthermore, top 10 circRNAs were selected for further verification by qRT-PCR by using designed divergent primers and found that only hsa_circ_0132015 and hsa_circ_0079474 were confirmed. We further found that hsa_circ_0079474 had more significant upregulation in IUA tissues and more abundance in IUA and normal tissues than hsa_circ_0132015 which was selected for further analysis (Fig. 1E). Furthermore, the expressions of E-cadherin and CK-18 were decreased while α-SMA and VIM were increased in TGF-β1-induced EECs than control EECs using qRT-PCR (Fig. 1F), Western blot (Fig. 1G) and immunocytochemistry (ICC) (Fig. 1H). The expression of hsa_circ_0079474 was also higher in TGF-β1-induced EECs than control EECs (Fig. 1I). Characteristics of hsa_circ_0079474 Analysis of circBase showed that hsa_circ_0079474 is originated from back splicing of exons 6-13 of human gene DGKB (also known as hsa_circ_DGKB-009) which was located on chromosome 7, with the length 812 nt (Fig. 2A). To confirm the results of the expression of hsa_circ_0079474 in IUA tissues, Sanger sequencing on the PCR products of hsa_circ_0079474 was performed. Sanger sequencing verified the back-splicing junction with the expected size and predicted splicing site in the PCR products (Fig. 2B). Agarose gel electrophoresis of reverse transcribed RNA (cDNA) and genomic DNA (gDNA) were performed by convergent and divergent primers for hsa_circ_­0079474. The results demonstrated that the divergent primers could amplify products from cDNA but not gDNA (Fig. 2C). As a confirmation of the stability of hsa_circ_0079474, the total RNA samples were treated with RNase R. The results of qRT-PCR displayed that the levels of linear DGKB (DGKB mRNA) was decreased significantly under RNase R treatment, whereas circDGKB-009 was resistant to the digestion of RNase R (Fig. 2D). The stability of hsa_circ_0079474 was further confirmed by treating EECs with Actinomycin D (a transcription inhibitor) for different periods of time. Results disclosed that the linear DGKB mRNA level decreased gradually with time but the level of circDGKB-009 was more stable and resistant to Actinomycin D administration (Fig. 2E). In addition, hsa_circ_0079474 was mainly located in cytoplasm measured by cytoplasmic and nuclear fractionation assay, with the positive control U6 in nucleus and GAPDH in cytoplasm, respectively (Fig. 2F). In brief, these findings indicated that hsa_circ_0079474 is a circRNA which is localized in the cytoplasm, has good stability, and may involve in the occurrence and development of IUA. Hsa_circ_0079474 promoted the progression of EMT To explore the functional roles of hsa_circ_0079474, gain-of-function and loss-of-function assays were performed, respectively. Firstly, overexpression plasmid of hsa_circ_0079474 was constructed and transfected to Ishikawa cells. The level of hsa_circ_0079474 was increased more than 60,000-fold in hsa_circ_0079474-overexpression of Ishikawa cells compared with control by qRT-PCR (Fig. 3A). Secondly, three specific siRNAs targeting hsa_circ_0079474 were transfected into Ishikawa cells to silence hsa_circ_0079474 expression. The results demonstrated that si-hsa_circ_0079474-3 had the highest efficiency and was selected for further functional trials (Fig. 3B). Subsequently, CCK-8 assays and Edu staining assays were conducted to determine the impact of hsa_circ_0079474 on the proliferation of Ishikawa cells. The findings disclosed that hsa_circ_0079474 overexpression led to improvement of the proliferation of Ishikawa cells whereas silence of hsa_circ_0079474 suppressed the proliferation of Ishikawa cells (Fig. 3C, D). Flow cytometry analysis elucidated that the up-regulation of hsa_circ_0079474 resulted in a decrease in the proportion of the G0/G1 phase but an increase in the percentage of the S phase of the cell cycle in Ishikawa cells. Conversely, down-regulation of hsa_circ_0079474 resulted in elevation in the percentage of the G0/G1 phase and a reduction in the proportion of the S phase of the cell cycle in Ishikawa cells, demonstrating that hsa_circ_0079474-overexpressing promoted cell cycle progression while down-regulation of hsa_circ_0079474 led to cell cycle arrest (Fig. 3E). Nevertheless, both up-regulation and down-regulation of hsa_circ_0079474 were found to have no significant impact on the proportion of G2/M phase of the cell cycle in Ishikawa cells. To further investigate the effects of hsa_circ_0079474 on EMT, qRT-PCR, Western blot and ICC were employed. The obtained results validated that overexpression of hsa_circ_0079474 led to a reduction in the levels of epithelial cell markers E-cadherin and CK-18 while increasing the levels of mesenchymal cell markers α-SMA and VIM. Conversely, knockdown of hsa_circ_0079474 yielded the opposite outcomes (Fig. 3F-H). In conclusion, hsa_circ_0079474 showed effects of promotion of cell proliferation, acceleration of cell cycle progression and improvement of EMT. Hsa_circ_0079474 served as a sponge for miR-630 It was postulated that hsa_circ_0079474 exerts its function through the mechanism of miRNA sponges, which is a prevalent mechanism for circRNAs. Circular RNA Interactome and CircBank datasets were employed to predict the potential binding miRNAs of hsa_circ_0079474. As exhibited in Venn diagram in Fig. 4A, three candidate miRNAs (hsa­-miR-630, hsa­-miR-892a and hsa­-miR-935) were identified to have putative binding sites on hsa_circ_0079474. The miRanda dataset was performed to suggest that miR-630, miR-892a and miR-935 could potentially bind to hsa_circ_0079474, with binding scores of 162, 156 and 151, and a total free energy of -14.39 kCal/Mol, -14.32 kCal/Mol and -16.67 kCal/Mol, respectively. These findings indicate that miR-630 is the most likely to bind hsa_circ_0079474. The expression levels of the aforementioned three miRNAs were subsequently assessed in both IUA and normal tissues. It was observed that miR-630 exhibited a significant decrease in IUA tissues compared to normal tissues whereas no significant differences were found in miR-892a and miR-935 (Fig. 4B). Additionally, the levels of miR-630 were determined in Ishikawa cells with overexpression of hsa_circ_0079474. The results indicated a significant decrease in miR-630 expression (Fig. 4C). Consequently, miR-630 was selected for further experimentation. The predicted binding sites of hsa_circ_0079474 to miR-630 were also displayed (Fig. 4D) and dual-luciferase reporter assay was performed to confirm the interaction between hsa_circ_0079474 and miR-630. The wild-type hsa_circ_0079474 sequences, which included potential miR-630 binding sites, as well as a mutation version, were inserted into the psiCHECK2 luciferase reporter. Results manifested that miR-630 remarkably inhibited the luciferase activity while miR-630 inhibitor notably enhanced luciferase activity in hsa_circ_0079474-wt reporter while not in the hsa_circ_0079474-mut reporter. This suggested that hsa_circ_0079474 has the ability to bind to miR-630 (Fig. 4E). Additionally, to understand the molecular mechanisms of hsa_circ_0079474 in IUA progression, subcellular localization was conducted in EECs by FISH. As presented in Fig. 4F. FISH results exhibited co-localization of miR-630 and hsa_circ_0079474 in the cytoplasm of EECs. RIP assays demonstrated that hsa_circ_0079474 was remarkably immunoprecipitated by Ago2 antibody in miR-630-transfected EECs compared to miR-NC-transfected EECs (Fig. 4G). These results confirmed that hsa_circ_0079474 served as a sponge for miR-630 in EECs. MiR-630 was downregulated in IUA and alleviated EMT process To further explore the involvement of miR-630 during IUA development, EECs and TGF-β1-induced EECs were collected and subjected to qRT-PCR to evaluate the expression of miR-630. Results manifested that the expression of miR-630 were lower in both IUA tissues and TGF-β1-induced EECs compared to normal tissues and EECs, respectively. (Fig. 5A). In addition, transfection of miR-630 mimics and miR-630 inhibitors into EECs confirmed the upregulation and downregulation of miR-630 (n=3) (Fig. 5B). Subsequently, CCK-8 and Edu staining assays were employed to observe the impacts of miR-630 on the property of proliferation of EECs. Results revealed that miR-630 inhibited the proliferation of EECs whereas suppression of miR-630 promoted the proliferation of EECs (Fig. 5C, D). Flow cytometry analysis further exhibited that miR-630 was found to have a restraining effect on the cell cycle while inhibition of miR-630 induced cell cycle progression (Fig. 5E). The results of expression of EMT markers proved that miR-630 enhanced the levels of epithelial cell markers (E-cadherin and CK-18) while decreasing the expression of mesenchymal cell markers (α-SMA and VIM) (Fig. 5F-H). In summary, miR-630 yielded properties of inhibition of cell proliferation, cell cycle progression and EMT. MiR-630 could directly target YAP1 To assess the potential target genes of miR-630, various datasets including miRDB, miRWalk, miRTarbase, RNACentral and TargetScan were utilized to predict. As a result, only one candidate gene (YAP1) was filtrated for further analysis (Fig. 6A). The two predictive binding sites on YAP1-3’UTR for miR-630 by miRanda were presented in Fig. 6B. To validate the binding between miR-630 and YAP1, dual-luciferase report assays was further conducted. Wild-type (Wt) YAP1-3’UTR and miR-630 binding site Mut luciferase reporter plasmids were transfected along with miR-630 mimic and miR-630 inhibitor. Results indicated that the relative luciferase activity of wt YAP1-3’UTR group was marked declined when transfected miR-630 mimic but remarkably increased when transfected miR-630 inhibitor. Conversely, no significantly differences of the relative luciferase activity were observed in mut YAP1-3’UTR groups (Fig. 6C). In addition, the expression of YAP1 was evaluated using qRT-PCR, Western blot and ICC following transfection with miR-630 mimic and miR-630 inhibitor and indicated that elevation of miR-630 dramatically inhibited the expression of YAP1 whereas downregulation of miR-630 distinctly improved the expression of YAP1 (Fig. 6D-F). These results confirmed that miR-630 could target YAP1 directly. YAP1 was increased in IUA and promoted EMT To investigate the underlying role of YAP1 in IUA development, samples of IUA tissues, normal tissues, EECs and TGF-β1-induced EECs were collected and subjected to qRT-PCR, Western blot and IHC/ICC analysis. Results concluded that the level of YAP1 in IUA tissues and TGF-β1-induced EECs was significantly higher than adjacent normal tissues and EECs respectively (Fig. 7A-C). To further explore the role of YAP1 in IUA development, YAP1-overexpression plasmid and siRNAs were transfected into EECs. The efficiency of transfection was confirmed using qRT-PCR. The transfection of YAP1-overexpression plasmid into EECs resulted in an increased level of YAP compared to the control group. (Fig. 7D). Next, three specific siRNAs targeting YAP1 were designed and transfected into EECs to silence YAP1 expression. It was illustrated that si-YAP1-3 exhibited the highest efficiency and was selected for further experiments (Fig. 7E). Further functional trials demonstrated that YAP1 overexpression resulted in a dramatically improvement of cell proliferation, cell cycle progression and EMT while knockdown of YAP1 disclosed opposite effects (Fig. 7F-K). Hsa_circ_0079474 promotes IUA progression via miR-630/YAP1 axis Furthermore, our study proposed to investigate the involvement of miR-630 in the regulation of hsa_circ_0079474 in Ishikawa cells. It was observed that the presence of hsa_circ_0079474 significantly enhanced cell proliferation, facilitated cell cycle progression, and promoted EMT. However, these effects were effectively counteracted by the restoration of miR-630 expression (Fig. 8A-F). Conversely, the depletion of hsa_circ_0079474 in Ishikawa cells was rescued by the administration of miR-630 inhibitor (Fig. 8G-L). The findings elucidated that hsa_circ_0079474 exerted its effects on Ishikawa cells, at least in part, by acting as a sponge for miR-630. Hsa_circ_0079474 promoted IUA via miR-630 in vivo The expression of E-cadherin and CK-18 were decreased while the expression of α-SMA and VIM increased compared with sham group by qRT-PCR and immunohistochemical staining, confirming that EMT was involved in the progression of IUA in rat (Fig.9A, B). Masson staining was applied to determine the degree of fibrosis. In IUA group, more fibrotic area was found than that of control group, suggesting that the IUA rat model was established successfully (Fig. 9B). It was further validated that IUA+AAV-hsa_circ_0079474 group notably decreased levels of E-cadherin and CK-18, accompanied by increased levels of α-SMA and VIM, as well as a greater presence of collagen fibers compared to the IUA group by qRT-PCR, IHC and Masson staining (Fig.9c-9d). Conversely, injection of si-hsa_circ_0079474 yielded contrasting results of AAV- hsa_circ_0079474 (Fig. 9C, D). In IUA+miR-630 agomir group, intrauterine injection of miR-630 agomir demonstrated higher levels of E-cadherin and CK-18 whereas lower levels of α-SMA and VIM, as well as lower rate of fibrotic area, which disclosed contrasting effects of hsa_circ_0079474 using qRT-PCR, IHC and Masson staining (Fig. 9E-F). Furthermore, administration of miR-630 antagomir exhibited opposite effects (Fig. 9E-F). However, these effects of AAV- hsa_circ_0079474 were at least partly reversed by administration of miR-630 agomir (Fig. 9G, H). Furthermore, the effects of hsa_circ_0079474-siRNA-chol were reversed at least partially by miR-630 antagomir (Fig. 9G, H). These findings displayed that hsa_circ_0079474 improved IUA via miR-630 in vivo . Discussion In recent years, emerging evidences have confirmed the roles of circRNAs in the development of multiple diseases, especially tumors. However, only three circRNAs have been reported thus far to be implicated in the progression of IUA. CircPlekha7 was first reported circRNA which played anti-fibrotic role in the IUA [ 10 ]. CircPTP4A2 contributed to endometrial repair progression by targeting miR-330-5p/PDK2 axis [ 11 ]. CircPTPN12 accelerated IUA progression by regulating miR-21-5 p/∆Np63α pathway [ 12 ]. In our study, we screened a new circRNA, hsa_circ_0079474, which was derived from the exons 6–13 of DGKB (also named circDGKB-009) by circRNA expression profiles. Although another circDGKB, hsa_circ_0133622 (circDGKB-017) was reported in the progression of neuroblastoma [ 16 ], the function of hsa_circ_0079474 has not been reported yet. We further confirmed the circular characteristics of hsa_circ_0079474 and revealed the mechanism of hsa_circ_0079474 in IUA development. It is proposed that the ceRNA mechanism for circRNAs, which means circRNAs might act as sponges of miRNAs to prevent miRNAs-triggered inhibition on the expression of target genes. In our present study, we investigated whether hsa_circ_0079474 exerted pro-fibrotic role in IUA by the above circRNA-miRNA-mRNA pathway. We confirmed that hsa_circ_0079474 was mainly located in the cytoplasm by FISH. Further bioinformatic analysis, luciferase reporter, and RIP assays validated that miR-630 was the target of hsa_circ_0079474 and was degraded by hsa_circ_0079474, as well as YAP1 was the target gene of miR-630. MiR-630 is a miRNA which encoded by MIR630 gene (NC_000015.10) on 15q24.1[ 17 ]. MiR-630 had been associated with multiple neoplastic and non-neoplastic conditions. The impacts of miR-630 on human malignancies had been fully elucidated but the results were controversial. MiR-630 represented anti-tumor properties in non-small cell lung cancer [ 18 ], esophageal cancer [ 19 ], thyroid cancer [ 20 ], cervical cancer [ 21 ] and breast cancer [ 22 ]. On the other hand, miR-630 acted as an oncogenic miRNA in colorectal cancer [ 23 ], ovarian cancer [ 24 ], renal cell cancer [ 25 ], prostate cancer [ 26 ] and acute lymphoblastic leukemia [ 27 ]. MiR-630 also had been involved in non-tumor disease such as IgA nephropathy [ 28 ] and cataract [ 29 ]. However, further studies were needed to investigate the potential role of miR-630 in fibrosis. In our experiment we examined the expression of miR-630 in IUA tissues and TGF-β1-induced EECs and proved that miR-630 yielded inhibition of IUA, contributing to a deeper understanding of its functional role. Several target genes of miR-630 had been validated by dual-luciferase reporter assay. For example, miR-630 could directly target vimentin, thus suppressing non-small cell lung cancer [ 18 ]. MiR-630 targeted IGF1R to regulate response to HER-targeting drugs and cancer progression in HER2 overexpressing breast cancer [ 22 ]. MiR-630 inhibited EMT by targeting Slug in hepatocellular carcinoma [ 30 ]. YAP1 had been previously confirmed as a target gene of miR-630 [ 31 – 33 ]. YAP1 had been involved in the progression of fibrosis including pulmonary fibrosis [ 34 ], cardiac fibrosis [ 35 ] and liver fibrosis [ 36 ]. In this experiment, we validated the binding of miR-630 and YAP1 by dual-luciferase reporter assay again and elucidated the effect of hsa_circ_0079474 on IUA via miR-630/YAP1 axis. In conclusion, hsa_circ_0079474 sponges miR-630 to facilitate YAP1 expression, thus improving EMT in IUA (Fig. 10 ). This finding provided a new insight into the molecular mechanism of circRNA in IUA and revealed that targeting hsa_circ_0079474/miR-630/YAP1 might be a promising therapeutic alternative for IUA. Materials and methods Tissues collection This study was authorized by the Ethics Committee of the First Affiliated Hospital with Nanjing Medical University (approval number 2022-SRFA-321). Paired fibrotic endometrial samples and adjacent normal endometrial samples were sampled from IUA patients who received hysteroscope at the First Affiliated Hospital with Nanjing Medical University. Informed consent was provided by all participants. We instantly frozen all samples in liquid nitrogen after hysteroscopy, and then stored them at -80°C until they were needed. For endometrial tissue collection, endometrial tissue biopsies were obtained from women with regular menstrual cycles and no exposure to steroids at least 3 months who received hysterectomy or subtotal hysterectomy for leiomyoma by curettage. CircRNA expression profiles Totally three paired IUA samples and adjacent normal samples were selected for circRNA expression profiles. The Agilent Human ceRNA Microarray 2019 was applicated in our study and further data analysis of all samples were performed by OE Biotechnology (Shanghai, China). Quantification of total RNA was performed using the NanoDrop ND-2000 (Thermo Scientific) and RNA integrity was evaluated by Agilent Bioanalyzer 2100 (Agilent Technologies). As directed by the manufacturer, samples were labeled, microarray hybridized, and washed in accordance with their instructions. Differentially expressed circRNAs were identified through fold change and P value calculated using the t-test. The threshold was a fold change ≥ 2.0 and a P value < 0.05. Cell culture and cell lines For primary endometrial epithelial cells (EECs) culture, endometria were instantly placed in culture medium and isolation began within 2 hours of collection. Endometria were cut up and digested in 1mg/ml collagenase I (Beijing Solarbio Science & Technology Co., Ltd) at 37°C for 60min. Following centrifugation, samples were filtered with a 150 nm strainer and a 38 nm strainer, respectively. The samples retained on the 38 nm strainer were regarded as EECs. The EECs were cultured in Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12) medium (Sigma-Aldrich). To induce EMT, TGF-β1(100ng/ml, Bioss) was added in the medium for EECs. Ishikawa cells and HEK-293T cells (Procell, Wuhan, China) were routinely cultured in DMEM medium with high glucose (KeyGEN, Nanjing, China). Both media contained 10% fetal bovine serum (Procell, Wuhan, China), 100U/ml penicillin and 0.1mg/ml streptomycin (Beyotime, Shanghai, China). Incubation was performed at 37°C in 5% CO2 and 95% air in a humidified atmosphere. Quantitative real-time polymerase chain reaction (qRT-PCR) An extraction of total RNA was carried out using Trizol reagent (Beyotime, Shanghai, China) from tissue samples and cells. The Nanodrop 2000 spectrophotometer (Thermo Fisher, USA) was used to measure the concentration and purity of the RNA samples. The cDNA was synthesized by HiScript III RT SuperMix for qPCR (+ gDNA wiper) (R323-01, Vazyme, Nanjing, China) for circRNAs and mRNAs. MiRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (MR101-01, Vazyme, Nanjing, China) was used for cDNA synthesis of miRNAs. The quantitative PCR was conducted by ChamQ SYBR qPCR Master Mix and miRNA Universal SYBR qPCR Master Mix (Q311-02 and MQ101-01, Vazyme, Nanjing, China) according the manufacturer’s instructions on an ABI 7500 RealTime PCR System (Applied Biosystems, USA). The level of miRNAs was normalized by U6, while circRNAs and mRNAs by glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The calculation of RNA expression was determined using the 2 −ΔΔCt method. The primers were synthesized by Tsingke Biotechnology Co., Ltd (Beijing, China) and listed in Additional file 1: Table S1 . Confirmation of circRNA To verify the circular characteristic of hsa_circ_0079474, the linear and circular transcripts of DGKB were amplified using convergent and divergent primers in both complementary DNA (cDNA) and genomic DNA (gDNA) isolated from IUA and adjacent normal samples. Agarose gel electrophoresis was applicated to separate the PCR products. Sanger sequencing was performed to confirm the sequence and back-splicing sites of hsa_circ_0079474. Actinomycin D and RNase R treatment To inhibit transcription, 2µg/ml Actinomycin D and dimethylsulfoxide (negative control) were added into the culture medium. For RNase R treatment, total RNA was subjected to 2U/µg RNase R (Geneseed, Guangzhou, China) and incubated at 37℃ for 20min. Following treatment, the RNAs were then subjected to reverse transcription using linear and circular DGKB primers for qRT-PCR. Isolation of cytoplasmic and nuclear RNA Cytoplasmic and nuclear fractionation assay was carried out by Nuclear and Cytoplasmic Extraction Kit (Beyotime, Shanghai, China) and Trizol reagent (Beyotime, Shanghai, China) in tissue samples following the guidelines provided by the manufacturer. Isolated cytoplasmic and nuclear RNA were then reverse transcribed by qRT-PCR to detect the relative expression of linear and circular DGKB. Construction of plasmid and cell transfection The sequences of hsa_circ_0079474 and YAP1 were amplified and subsequently inserted into pLC5-ciR vector (Geneseed, Guangzhou, China) and pENTER vector (Vigene, Ji’nan, China) to construct hsa_circ_0079474 or YAP1-overexpressing plasmid, respectively. Three small interfering RNAs (siRNAs) targeting the junction sequence of hsa_circ_0079474 (si-hsa_circ_0079474-1, forward 5’-UACUGGUCUGAGAAUGAAUTT-3’ and reverse 5’-AUUCAUUCUCAGACCAGUATT − 3’; si-hsa_circ_0079474-2, forward 5’-AGUGGUACUGGUCUGAGAATT-3’ and reverse 5’-UUCUCAGACCAGUACCACUTT-3’; si-hsa_circ_0079474-3, forward 5’- GGUACUGGUCUGAGAAUGATT-3’ and reverse 5’-UCAUUCUCAGACCAGUACCTT-3’), YAP1 (si-YAP1-1, forward 5’-GGUGAUACUAUCAACCAAATT-3’ and reverse 5’-UUUGGUUGAUAGUAUCACCTT-3’; si-YAP1-2, forward 5’- CAUUAACGACUAGAUUAAATT-3’ and reverse 5’- UUUAAUCUAGUCGUUAAUGTT-3’; si-YAP1-3 forward 5’- GCCACAGAUUAAGAUUAUATT-3’ and reverse 5’- UAUAAUCUUAAUCUGUGGCTT-3’) and control non-specific oligonucleotides (si-NC, forward 5’- UUCUCCGAACGUGUCACGUTT-3’ and reverse 5’- ACGUGACACGUUCGGAGAATT-3’) were obtained from Vigene (Ji’nan, China). MiR-630 mimics, miR-630 inhibitor and scrambled control (miR-NC) were synthesized by Sangon (Shanghai, China). Above siRNAs and miRNAs were transfected into Ishikawa cells by application of Lipo8000™ Transfection Reagent (Beyotime, Shanghai, China) according to the manual of manufacturer. Cell counting kit-8 (CCK-8) assay CCK-8 assay was conducted to evaluate the proliferation of Ishikawa cells. Cells were seeded and cultured in 96-well plates for 0, 24h, 48h, 72h and 96h. CCK-8 solution (New Cell & Molecular Biotech, Suzhou, China) was added to each well (10µl/plates). The absorbance at 450 nm was measured by a spectrophotometer (Thermo Scientific) after 1 h of incubation at 37°C. 5-Ethynyl-2 ’ -Deoxyuridine (EdU) assay The EdU assay was conducted with a EdU Cell Proliferation Kit (Beyotime, Shanghai, China). The Ishikawa cells were fixed in 4% paraformaldehyde after being incubated for 2 hours with 10µM EdU. Hoechst-33,342 (Beyotime, Shanghai, China) was applicated to stain the nucleus. An EdU-positive cell percentage was calculated from images acquired with a fluorescence microscope (Nikon, Japan). Cell cycle assay Transfected Ishikawa cells were fixed in 70% ethanol at 4°C overnight and stained with propidium iodide (Beyotime, Shanghai, China). Cell cycle analysis was performed by flow cytometer (BD FACSCanto, USA). The ratio of cells in different phase was counted and compared. Dual-luciferase reporter assay To validate the binding of hsa_circ_0079474 and miR-630, as well as miR-630 and YAP1, dual-luciferase reporter assay was conducted. Wild type (WT) and mutant (MUT) sequence of hsa_circ_0079474 and YAP1 of predicted binding sites with miR-630 were synthesized and cloned into psiCHECK2 reporter vector. The reporter vector, miR-630 mimics, miR-630 inhibitor and negative control were transfected in HEK-293T cells by Lipofectamine 2000 (Invitrogen, USA). Activity of firefly and renilla luciferase were evaluated by TransDetect ® Double-Luciferase Reporter Assay Kit (TransGen, Beijing, China) in accordance to the manufacture’s manual. Fluorescence in situ hybridization (FISH) Hybridization was conducted overnight at 37°C with specific probes with fluorescence label of hsa_circ_0079474 and miR-630. 4',6-diamidino-2-phenylindole (DAPI) was counterstained for 20 min to stain nucleus. The sections were observed under a fluorescence microscope (Nikon, Japan). Nuclei stained blue while hsa_circ_0079474 which was labeled with Cy3 stained red, and miR-630 which was labeled with fluorescein isothiocyanate (FITC) stained green. FISH probes were sequenced as follows.: hsa_circ_0079474, 5′-tattcattctcagaccagtaccact-3′; miR-630, 5′- AGUAUUCUGUACCAGGGAAGGU-3′; Both the probes were synthesized by Geneseed (Guangzhou, China). Bioinformatic analysis The binding between hsa_circ_0079474 and miR-630 was predicted by Circular RNA Interactome ( https://circinteractome.nia.nih.gov/ ) and CircBank ( http://www.circbank.cn/ ) while the interaction between miR-630 and YAP1 was predicted by TargetScan ( https://www.targetscan.org/vert_71/ ), miRTarbase ( https://mirtarbase.cuhk.edu.cn/php/index.php ), miRCentral ( https://rnacentral.org/ ), miRDB ( http://mirdb.org/ ) and miRWalk ( http://mirwalk.umm.uni-heidelberg.de/ ). RNA immunoprecipitation (RIP) assay The RIP assay was performed by RIP Kit (Geneseed, Guangzhou, China) in accordance to the instructions of the manufacturer. Briefly, Ishikawa cells were collected, lysed and Ago2 antibodies (normal control) or IgG (Ishikawa cells) were conjugated with magnetic beads and incubated overnight at 4°C. After washing the beads, proteinase K was used to dissolve proteins. An agarose gel electrophoresis and quantitative RT-PCR were conducted after RNA extraction. Western blotting Total proteins from cells and tissues were extracted by RIPA Lysis Buffer (Beyotime, Shanghai, China) and the concentration of protein were performed by BCA protein assay kit (Bioss, Beijing, China). Proteins were separated and resolved in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred into nitrocellulose membranes and then subjected to blocking of skim milk and incubation of primary antibodies of E-cadherin (AF6759, Beyotime, Shanghai, China), CK-18 (CSB-MA000253, CUSABIO, https://www.cusabio.com/ ), α-SMA (ab5694, Abcam, UK), Vimentin (ab8978, Abcam, UK), YAP1(bs-3605R, Bioss, Beijing, China), GAPDH (AF0006, Beyotime, Shanghai, China) and horseradish peroxidase-conjugated secondary goat anti-mouse/rabbit antibody (Beyotime, Shanghai, China). Animal experiments in vivo For establishment of IUA model, 8-week-old female Sprague-Dawley rats (Animal Core Facility of Nanjing Medical University) were utilized. Rats were housed under a 12h:12h light-dark cycle with free access to water and food. The protocols of the experiment were conducted according to the guidelines of Institutional Animal Care and Use of Laboratory Animal and authorized by the Laboratory Animal Ethics Committee of Nanjing Medical University (approved number: IACUC-1912051). The rat model of IUA was conducted by endometrial scraping in accordance to our method which previous reported [ 16 ]. Briefly, rats were anesthetized by intraperitoneal injection of pentobarbital (40 mg/kg). Rats were divided into eight groups (n = 3 per group): sham-operation group, IUA group, IUA + adeno-associated virus (AAV)-hsa_circ_0079474 group, IUA + si-79474 group, IUA + miR-630 agomir group, IUA + miR-630 antagomir group, IUA + AAV-hsa_circ_0079474 + miR-630 agomir group and IUA + si-79474 + miR-630 antagomir group. For sham-operation group, the abdomen was cut and sutured without treating the uterus. For other groups, after the Y‑type uterus was exposed, the bilateral uterus was cut and scraped by a razor blade. The wound was then sutured for IUA group. Intrauterine injection of AAV-hsa_circ_0079474 (OBiO Technology Corp., Ltd, Shanghai, China) (1*10^10 viral genomes per rat), miR-630 agomir (5nmol per rat), miR-630 antagomir (10nmol per rat) and hsa_circ_0079474-siRNA-chol (10nmol per rat) were utilized in the left uterus of rats while AAV-vector, agomir-NC (5nmol per rat) antagomir-NC (10nmol per rat) and siRNA-NC (10nmol per rat) were injected in the right uterus of the corresponding rat. Rats were sacrificed by intraperitoneal injection of pentobarbital (100 mg/kg,) and uteri were cut and collected 14 days after surgery. For histological evaluation, tissues were fixed in 4% paraformaldehyde, embedded in paraffin for cutting slices. Masson staining was utilized to determine the degree of fibrosis. Immunohistochemical staining For immunohistochemical (IHC) staining, paraffin sections were firstly deparaffinized and rehydrated. For immunocytochemistry (ICC), samples were first fixed in formalin for 20min. Sections/Samples were then immersed into 0.3% Triton X-100. Antigen retrieval was performed with 0.1 mmol/L sodium citrate at 95–100°C for 15 minutes, followed by incubation in 3% hydrogen peroxide to block endogenous peroxidase activity. Primary antibodies (listed in the Western blotting) were incubated overnight at 4°C then incubated with secondary antibody. Sections were then stained with 3,3’-diaminobenzidine (ZhongShan Golden Bridge, Beijing, China) for visualization. Statistical analysis All data were presented as mean ± standard deviation and analyzed by SPSS 24.0. Comparisons between two groups were performed by Student’s t test. while three or more groups were conducted by one-way analysis of variance (ANOVA) followed by Dunnett’s test. P < 0.05 were regarded as statistical differences while P < 0.01 were considered as significant statistical differences. Declarations Acknowledgements Fig.10 was drawn by Figdraw. Thank you for pathologists of the First Affiliated Hospital with Nanjing Medical University (Yang Li and Guoqiang Ping) for preparing tissue sections. Funding This study was supported by the Young Scholars Fostering Fund of the First Affiliated Hospital of Nanjing Medical University (PY2021003). Availability of data and materials The data used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. References Westendorp IC, Ankum WM, Mol BW, Vonk J. Prevalence of Asherman’s syndrome after secondary removal of placental remnants or a repeat curettage for incomplete abortion. Hum Reprod. 1998;13(12):3347-50. Salzani A, Yela DA, Gabiatti JRE, Bedone AJ, Monteiro IMU. 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Additional Declarations There is no duality of interest Supplementary Files TableS1.docx Table S1 Figure1Bwestern.pdf Figure 1B-western Figure1Gwestern.pdf Figure 1G-western Figure3Gwestern.pdf Figure5Gwestern.pdf Figure6Ewestern.pdf Figure 6E-western Figure7Bwestern.pdf Figure 7B-western Figure7Jwestern.pdf Figure 7J-western Figure8Bwestern.pdf Figure 8B-western Figure8Hwestern.pdf Figure 8H-western Cite Share Download PDF Status: Under Review 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. 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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-3767908","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":264814894,"identity":"a87c4a7a-9326-43e6-b151-4f390dfc0455","order_by":0,"name":"Chen 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1","display":"","copyAsset":false,"role":"figure","size":13835233,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of epithelial-mesenchymal transition (EMT) markers and circRNA expression profiles in intrauterine adhesion (IUA).\u003c/strong\u003e \u003cstrong\u003eA-C \u003c/strong\u003eThe expression of\u003cstrong\u003e \u003c/strong\u003eEMT markers including epithelial cell markers (E-cadherin and CK-18) and mesenchymal cell markers (α-SMA and VIM) were evaluated using qRT-PCR(n=6) (\u003cstrong\u003eA\u003c/strong\u003e), Western blot (n=6) (\u003cstrong\u003eB\u003c/strong\u003e) and immunohistochemistry (IHC) (\u003cstrong\u003eC\u003c/strong\u003e) in both IUA tissues and adjacent non-IUA tissues.\u003cstrong\u003e D \u003c/strong\u003eA heatmap demonstrated different expressed circRNAs in both IUA tissues and adjacent non-IUA tissues. \u003cstrong\u003eE\u003c/strong\u003eThe expression of hsa_circ_0132015 and hsa_circ_0079474 were measured in both IUA tissues and adjacent non-IUA tissues by qRT-PCR (n=9). \u003cstrong\u003eF-H\u003c/strong\u003e The levels of epithelial cell markers (E-cadherin and CK-18) and mesenchymal cell markers (α-SMA and VIM) in endometrial epithelial cells (EECs) and TGF-β1-induced EECs by qRT-PCR (n=6) (\u003cstrong\u003eF\u003c/strong\u003e), Western blot (\u003cstrong\u003eG\u003c/strong\u003e) and immunocytochemistry (ICC) (\u003cstrong\u003eH\u003c/strong\u003e). \u003cstrong\u003ei\u003c/strong\u003e The expression of hsa_circ_0079474 were determined in EECs and TGF-β1-induced EECs by qRT-PCR (n=9) (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001). Bar=50μm.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3767908/v1/f3e1f71eb3f81bad1e1cee85.jpg"},{"id":50169795,"identity":"cfaf2911-aed0-4ffb-9d17-de94eec7dc47","added_by":"auto","created_at":"2024-01-25 15:33:56","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4195616,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacteristics of hsa_circ_0079474.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e The schematic diagram demonstrates that hsa_circ_0079474 is derived from head-to-tail splicing from DGKB gene, ranging from the 6th exon to the 13th exon. \u003cstrong\u003eB\u003c/strong\u003e The result of Sanger sequencing verified the back-splicing junction and predicted splicing site in the PCR products. \u003cstrong\u003eC\u003c/strong\u003e Agarose gel electrophoresis of reverse transcribed RNA (cDNA) and genomic DNA (gDNA) by convergent primers and divergent primers for hsa_circ_0079474. \u003cstrong\u003eD, E\u003c/strong\u003e The levels of linear DGKB (DGKB mRNA) and circDGKB-009 (hsa_circ_0079474) were measured under RNase R (n=6) (\u003cstrong\u003eD\u003c/strong\u003e) and Actinomycin D (\u003cstrong\u003eE\u003c/strong\u003e) treatment. \u003cstrong\u003eF\u003c/strong\u003e The expression level of hsa_circ_0079474 in the nuclear and cytoplasmic fractions of EECs using qRT-PCR (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3767908/v1/86f503c4485fb5d6137a4595.jpg"},{"id":50170947,"identity":"6354415c-ef4a-45f7-90fd-140bdb3992a1","added_by":"auto","created_at":"2024-01-25 15:41:57","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":23428125,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe function of hsa_circ_0079474. A, B \u003c/strong\u003eGain-of-function and loss-of-function assays were conducted using qRT-PCR to observe the transfection efficiency of hsa_circ_0079474-overexpressing\u003cstrong\u003e \u003c/strong\u003eplasmid in Ishikawa cells (\u003cstrong\u003eA\u003c/strong\u003e) and siRNAs targeting hsa_circ_0079474 (si-circ #1, si-circ #2 and si-circ #3) in Ishikawa cells (\u003cstrong\u003eB\u003c/strong\u003e) (n=3). \u003cstrong\u003eC-E\u003c/strong\u003e CCK-8 (n=3) (\u003cstrong\u003eC\u003c/strong\u003e), Edu staining (n=4) (\u003cstrong\u003eD\u003c/strong\u003e) and flow cytometry (\u003cstrong\u003eE\u003c/strong\u003e) assays were utilized to assess the influences of hsa_circ_0079474 overexpression and knockdown on cell proliferation and cell cycle distribution. \u003cstrong\u003eF-H \u003c/strong\u003eThe levels of epithelial-mesenchymal transition (EMT) markers E-cadherin, CK-18, α-SMA and VIM following transfection of hsa_circ_0079474-overexpressing\u003cstrong\u003e \u003c/strong\u003eplasmid (n=4) and siRNA (n=3) targeting hsa_circ_0079474 in Ishikawa cells by qRT-PCR (\u003cstrong\u003eF\u003c/strong\u003e), Western blot (\u003cstrong\u003eG\u003c/strong\u003e) and immunocytochemistry (ICC) (\u003cstrong\u003eH\u003c/strong\u003e) (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001). Bar=50μm.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3767908/v1/2dee73137f042013cac77830.jpg"},{"id":50169801,"identity":"4e5c5003-2cb2-445c-a2f9-30b2060b0ee5","added_by":"auto","created_at":"2024-01-25 15:33:57","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5110139,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHsa_circ_0079474 served as a sponge for miR-630.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003eVenn diagram exhibiting the overlapping of Circular RNA Interactome and CircBank datasets to predict the miRNAs with potential binding abilities of hsa_circ_0079474. \u003cstrong\u003eB\u003c/strong\u003e The expression of miR-630, miR-892a, and miR-935 in both IUA tissues and adjacent non-IUA tissues by qRT-PCR (n=6). \u003cstrong\u003eC\u003c/strong\u003e The levels of miR-630 with administration of hsa_circ_0079474 overexpression plasmid in Ishikawa cells by qRT-PCR (n=4). \u003cstrong\u003eD\u003c/strong\u003e The predicted binding sites of hsa_circ_0079474 to miR-630. \u003cstrong\u003eE\u003c/strong\u003e The luciferase activities of hsa_circ_0079474-wt and hsa_circ_0079474-mut reporters in Ishikawa cells transfected with mimic NC, miR-630 mimic, inhibitor NC and miR-630 inhibitor were determined by dual-luciferase reporter assays (n=3). \u003cstrong\u003eF\u003c/strong\u003e Colocalization between hsa_circ_0079474 and miR-630 by FISH in Ishikawa cells. Nuclei were stained with DAPI. \u003cstrong\u003eG\u003c/strong\u003e RIP assays were performed using Ago2 or IgG antibody in Ishikawa cells transfected with miR-630 and hsa_circ_0079474 (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001). Bar=50μm.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3767908/v1/b8146025c13684d23a34ac22.jpg"},{"id":50169810,"identity":"cd5806a8-9bfa-461c-939b-59d3be5c649b","added_by":"auto","created_at":"2024-01-25 15:33:57","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":23125927,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe function of miR-630.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e The expression of miR-630 in both endometrial epithelial cells (EECs) and TGF-β1-induced EECs by qRT-PCR (n=10). \u003cstrong\u003eB\u003c/strong\u003eThe expression of miR-630 were performed using qRT-PCR to observe the transfection efficiency of administration of miR-630 mimic and miR-630 inhibitor (n=3). \u003cstrong\u003eC-E\u003c/strong\u003e CCK-8 (n=3) (\u003cstrong\u003eC\u003c/strong\u003e), Edu staining (n=4) (\u003cstrong\u003eD\u003c/strong\u003e) and flow cytometry (\u003cstrong\u003eE\u003c/strong\u003e) assays were performed to investigate the effects of administration of miR-630 mimic and miR-630 inhibitor on cell proliferation and cell cycle distribution. \u003cstrong\u003eF-H \u003c/strong\u003eThe expression of E-cadherin, CK-18, α-SMA and VIM after transfection of miR-630 mimic and miR-630 inhibitorin Ishikawa cells by qRT-PCR (n=3) (\u003cstrong\u003eF\u003c/strong\u003e), Western blot (\u003cstrong\u003eG\u003c/strong\u003e) and immunocytochemistry (ICC) (\u003cstrong\u003eH\u003c/strong\u003e) (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001). Bar=50μm.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3767908/v1/078c593f1cb7d7a93c916273.jpg"},{"id":50169803,"identity":"9f87fb66-4a68-44dd-b7a3-e12760db94d3","added_by":"auto","created_at":"2024-01-25 15:33:57","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6352266,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eYAP1 is a target gene for miR-630.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003eVeen diagram demonstrating the prediction of target genes of miR-630 according to various datasets. \u003cstrong\u003eB\u003c/strong\u003e The predicted binding sites of miR-630 to YAP1. \u003cstrong\u003eC\u003c/strong\u003e The luciferase activities of YAP1-wt and YAP1-mut reporters transfected with mimic NC, miR-630 mimic, inhibitor NC and miR-630 inhibitor were determined by dual-luciferase reporter assays in Ishikawa cells(n=3). \u003cstrong\u003eD-F \u003c/strong\u003eThe expression of YAP1 followed by transfection of miR-630 mimic and miR-630 inhibitor in Ishikawa cells by qRT-PCR (n=3) (\u003cstrong\u003eD\u003c/strong\u003e), Western blot (\u003cstrong\u003eE\u003c/strong\u003e) and immunocytochemistry (ICC) (\u003cstrong\u003eF\u003c/strong\u003e) (*p\u0026lt;0.05, ***p\u0026lt;0.001). Bar=50μm.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3767908/v1/96dcd88cee7bd9b6ad02224d.jpg"},{"id":50169809,"identity":"55859416-9d0a-49c8-9d7f-0edf6856bf20","added_by":"auto","created_at":"2024-01-25 15:33:57","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":27815781,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe function of YAP1. A-C \u003c/strong\u003eThe expression of YAP1 were evaluated in both IUA tissues, adjacent non-IUA tissues, endometrial epithelial cells (EECs) and TGF-β1-induced EECs by qRT-PCR (n=9) (\u003cstrong\u003eA\u003c/strong\u003e), Western blot (\u003cstrong\u003eB\u003c/strong\u003e) and immunohistochemistry/immunocytochemistry (IHC/ICC) (\u003cstrong\u003eC\u003c/strong\u003e). \u003cstrong\u003eD, E \u003c/strong\u003eqRT-PCR was conducted to investigate the efficiency of transfection using YAP1-overexpressing\u003cstrong\u003e \u003c/strong\u003eplasmid (\u003cstrong\u003eD\u003c/strong\u003e) and siRNAs targeting YAP1(si-YAP1 #1, si-YAP1 #2 and si-YAP1 #3) in Ishikawa cells (\u003cstrong\u003eE\u003c/strong\u003e) (n=3). \u003cstrong\u003eF-H \u003c/strong\u003eCCK-8 (n=3) (\u003cstrong\u003eF\u003c/strong\u003e), Edu staining (n=4) (\u003cstrong\u003eG\u003c/strong\u003e) and flow cytometry (\u003cstrong\u003eH\u003c/strong\u003e) assays were used to evaluated the impacts of YAP1-overexpression plasmid and si-YAP1 on cell proliferation and cell cycle. \u003cstrong\u003eI-K \u003c/strong\u003eEffects of transfection of YAP1-overexpressing\u003cstrong\u003e \u003c/strong\u003eplasmid (n=4) and siRNA (n=3) targeting YAP1 on the expression of EMT markers (E-cadherin, CK-18, α-SMA and VIM) by qRT-PCR (\u003cstrong\u003eI\u003c/strong\u003e), Western blot (\u003cstrong\u003eJ\u003c/strong\u003e) and immunocytochemistry (ICC) (\u003cstrong\u003eK\u003c/strong\u003e) in Ishikawa cells (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001). Bar=50μm.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3767908/v1/9cc6d732ae0bfb94c244d39f.jpg"},{"id":50169811,"identity":"84f25ba6-fab4-457c-90c0-8b56ade8176a","added_by":"auto","created_at":"2024-01-25 15:33:57","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":15954276,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHsa_circ_0079474 promotes IUA progression via miR-630.\u003c/strong\u003e \u003cstrong\u003eA-F\u003c/strong\u003e The effects of hsa_circ_0079474 on the expression of YAP1 and epithelial-mesenchymal transition (EMT) marker, cell proliferation and cell cycle distribution were counteracted by administration of miR-630 mimic by qRT-PCR (n=3) (*p\u0026lt;0.05) (\u003cstrong\u003eA\u003c/strong\u003e), Western blot (\u003cstrong\u003eB\u003c/strong\u003e), immunocytochemistry (ICC) (\u003cstrong\u003eC\u003c/strong\u003e), CCK-8 (\u003cstrong\u003eD\u003c/strong\u003e), Edu staining (\u003cstrong\u003eE\u003c/strong\u003e) and flow cytometry (\u003cstrong\u003eF\u003c/strong\u003e).\u003cstrong\u003e G-L\u003c/strong\u003e The effects of si-hsa_circ_0079474 on the expression of YAP1 and epithelial-mesenchymal transition (EMT) marker, cell proliferation and cell cycle distribution were counteracted by administration of miR-630 inhibitor, as determined by qRT-PCR (n=3) (*p\u0026lt;0.05) (\u003cstrong\u003eG\u003c/strong\u003e), Western blot (\u003cstrong\u003eH\u003c/strong\u003e), immunocytochemistry (ICC) (\u003cstrong\u003eI\u003c/strong\u003e), CCK-8 (\u003cstrong\u003eJ\u003c/strong\u003e), Edu staining (\u003cstrong\u003eK\u003c/strong\u003e) and flow cytometry (\u003cstrong\u003eL\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3767908/v1/09329f0284246074c8be24ff.jpg"},{"id":50169806,"identity":"0b72e132-7091-4d38-bfc7-a6aa411c58af","added_by":"auto","created_at":"2024-01-25 15:33:57","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":8028908,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHsa_circ_0079474 promoted IUA via miR-630 \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo.\u003c/strong\u003e\u003c/em\u003e \u003cstrong\u003eA-B\u003c/strong\u003e\u003cem\u003e \u003c/em\u003eThe expression of E-cadherin, CK-18, α-SMA and VIM by qRT-PCR \u003cstrong\u003e(A)\u003c/strong\u003e and immunohistochemistry and Masson staining \u003cstrong\u003e(B)\u003c/strong\u003e in sham and IUA group. \u003cstrong\u003eC-D\u003c/strong\u003e The levels of epithelial-mesenchymal transition (EMT) markers E-cadherin, CK-18, α-SMA, VIM and fibrotic rate of uteri following injection of AAV-hsa_circ_0079474 and hsa_circ_0079474-siRNA-chol by qRT-PCR \u003cstrong\u003e(C)\u003c/strong\u003e, immunohistochemistry (IHC) and Masson staining \u003cstrong\u003e(D)\u003c/strong\u003e. \u003cstrong\u003eE-F\u003c/strong\u003e The levels of epithelial-mesenchymal transition (EMT) markers E-cadherin, CK-18, α-SMA, VIM and fibrotic rate of uteri following injection of miR-630 agomir and miR-630 antagomir by qRT-PCR \u003cstrong\u003e(E)\u003c/strong\u003e, immunohistochemistry (IHC) Masson staining \u003cstrong\u003e(F)\u003c/strong\u003e.\u003cstrong\u003e G-H \u003c/strong\u003eThe effects of AAV-hsa_circ_0079474 and hsa_circ_0079474-siRNA-chol on the expression of EMT markers E-cadherin, CK-18, α-SMA, VIM and fibrotic rate of uteri were restored by administration of miR-630 agomir and miR-630 antagomir by qRT-PCR (\u003cstrong\u003eG\u003c/strong\u003e), immunohistochemistry (IHC) and Masson staining \u003cstrong\u003e(H)\u003c/strong\u003e, respectively (**p\u0026lt;0.01, *p\u0026lt;0.05). Bar=50μm.\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3767908/v1/8810a17455e3f46836a5f874.jpg"},{"id":50169804,"identity":"1f34617c-5496-4cea-a706-8682874724db","added_by":"auto","created_at":"2024-01-25 15:33:57","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":3438292,"visible":true,"origin":"","legend":"\u003cp\u003eThe mechanism diagram was generated to illustrate the mechanism of hsa_circ_0079474/miR-630/YAP1 axis in the development of IUA.\u003c/p\u003e","description":"","filename":"Figure10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3767908/v1/26439b6490bf87b26bf70f3a.jpg"},{"id":50172738,"identity":"260cc54e-de7f-42d2-a82b-270040b3bbbd","added_by":"auto","created_at":"2024-01-25 15:58:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2281844,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3767908/v1/55bbd723-32c9-4f47-ad2d-ec7d345e7fc3.pdf"},{"id":50169791,"identity":"539371dd-5cdc-443e-92b0-12f872afb704","added_by":"auto","created_at":"2024-01-25 15:33:56","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17627,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1\u003c/p\u003e","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3767908/v1/8a06071c638186ddfdcafa26.docx"},{"id":50170941,"identity":"6395416e-d04b-43ca-94e3-a1e1973527e5","added_by":"auto","created_at":"2024-01-25 15:41:56","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":167896,"visible":true,"origin":"","legend":"Figure 1B-western","description":"","filename":"Figure1Bwestern.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3767908/v1/fe35e09b6d188a287bfcb6c2.pdf"},{"id":50169792,"identity":"8b259f0d-9fb6-4cc4-8641-b5505d0aa4c2","added_by":"auto","created_at":"2024-01-25 15:33:56","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":330341,"visible":true,"origin":"","legend":"Figure 1G-western","description":"","filename":"Figure1Gwestern.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3767908/v1/e4c53df0998648d35916011c.pdf"},{"id":50170945,"identity":"d84bd232-de55-4696-a6bd-109b63ab0104","added_by":"auto","created_at":"2024-01-25 15:41:57","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":688680,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3Gwestern.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3767908/v1/2cc08e0da598df3e451b73d9.pdf"},{"id":50171929,"identity":"76f24cb6-7072-42c8-adc0-3d83b53df825","added_by":"auto","created_at":"2024-01-25 15:49:57","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":634808,"visible":true,"origin":"","legend":"","description":"","filename":"Figure5Gwestern.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3767908/v1/baa98de767fcdeee6702af99.pdf"},{"id":50169797,"identity":"88a34b72-79ab-46dd-bbac-380bbda8f49d","added_by":"auto","created_at":"2024-01-25 15:33:56","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":178139,"visible":true,"origin":"","legend":"Figure 6E-western","description":"","filename":"Figure6Ewestern.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3767908/v1/bc024b332046a5a4c889ffca.pdf"},{"id":50170942,"identity":"08117dcf-3a60-476d-a771-39af18a9f0f7","added_by":"auto","created_at":"2024-01-25 15:41:56","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":150102,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 7B-western\u003c/p\u003e","description":"","filename":"Figure7Bwestern.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3767908/v1/e5adf12c32071f57341ea6b6.pdf"},{"id":50169799,"identity":"2020b6a2-eab2-48c8-8c05-3044ff4bd995","added_by":"auto","created_at":"2024-01-25 15:33:56","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":635161,"visible":true,"origin":"","legend":"Figure 7J-western","description":"","filename":"Figure7Jwestern.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3767908/v1/c0c8cc62a63f906ef51a515e.pdf"},{"id":50170946,"identity":"1824e9ec-9a7b-45bf-810a-f12118758143","added_by":"auto","created_at":"2024-01-25 15:41:57","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":369445,"visible":true,"origin":"","legend":"Figure 8B-western","description":"","filename":"Figure8Bwestern.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3767908/v1/4b1c1f91ffd677e3d83a171e.pdf"},{"id":50170948,"identity":"943702fb-5695-4779-b523-afcd9c884817","added_by":"auto","created_at":"2024-01-25 15:41:57","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":392251,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 8H-western\u003c/p\u003e","description":"","filename":"Figure8Hwestern.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3767908/v1/7f771be6a2c559f6a23d68f7.pdf"}],"financialInterests":"There is no duality of interest","formattedTitle":"Hsa_circ_0079474 facilitates epithelial-mesenchymal transition in intrauterine adhesion via miR-630/YAP1 axis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIntrauterine adhesion (IUA) is characterized by the development of endometrial fibrosis in uterine cavity and cervix which is derived from damage to the basal layer of the endometrium. With the rapid growth of artificial abortion, curettage and hysteroscopic operations, the incidence of IUA is explosive growing. Approximately 40% of females with incomplete abortions who experienced curettage developed IUA [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Since most cases are asymptomatic, predicting the true prevalence of IUA is difficult. A variety of treatments including hysteroscopy, intrauterine devices and hormonal therapies have been applied in ameliorating IUA, but showed limited effects for women with moderate and severe IUA especially, which also had high recurrence rate [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, it is essential to better understand the mechanisms of IUA and explore more alternative therapies to improve the outcome of menstruation and pregnancy.\u003c/p\u003e \u003cp\u003eEpithelial-mesenchymal transition (EMT) is a biological phenomenon involving epithelial cells losing their typical epithelial phenotype and acquiring mesenchymal characteristics such as proliferative, migratory and invasive properties. EMT is involved in various pathophysiological process including embryogenesis, tissue construction, formation of fibrosis and cancer metastasis. EMT is identified by reduced levels of epithelial cell markers like E-cadherin and cytokeratin, accompanied by elevated levels of mesenchymal cell markers such as alpha smooth muscle actin (α-SMA) and Vimentin (VIM) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. EMT had also been observed playing an important role in the pathogenesis of endometrial fibrosis during IUA [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, EMT remains to be studied further in order to determine the exact mechanisms involved in IUA development.\u003c/p\u003e \u003cp\u003eCircular RNAs (circRNAs) are a subclass of non-coding RNAs which are characterized by forming a covalently closed loop structure, resulting resistance to exonuclease digestion [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Recently, circRNAs have been implicated in multiple pathophysiological processes in numerous studies. However, only circPlekha7 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], circPTP4A2 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and circPTPN12 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] were reported to be involved in IUA. There is still a lack of understanding regarding the molecular mechanisms underlying circRNAs in the process of IUA.\u003c/p\u003e \u003cp\u003eMicroRNAs (miRNAs) are another type of non-coding RNAs which consist of 20\u0026ndash;23 nucleotides. MiRNAs take participate in various biological process via negative regulation of target genes by binding to complementary sequences within mRNA targets at the level of post-transcription [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. CircRNAs have miRNA response elements and may play regulatory role by sponging miRNAs to neutralize miRNA-mediated effects on downstream mRNAs, which is the main regulatory mechanisms of circRNAs in the numerous physiological and pathological processes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the current research, we examined a screening of circRNA expression patterns in IUA tissues and normal endometrium using a circRNA microarray. Following filtration and verification, we confirmed that hsa_circ_0079474, a circRNA originated from the exons 6\u0026ndash;13 of human gene DGKB (diacylglycerol kinase beta), which was also known as circDGKB-009, exhibited significantly higher expression levels in IUA tissues compared to normal endometrium. Subsequently, we delved into the mechanism by which hsa_circ_0079474 contributes to the development of IUA and discovered that it exerts fibrotic effects by acting as a miR-630 sponge to activate its target gene yes-associated protein-1 (YAP1). These findings provided novel insights into circRNA-mediated IUA regulation and exploring new promising biomarkers for diagnosis of IUA.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eHsa_circ_0079474 is upregulated in IUA patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSince EMT is a pivotal event in the progression of fibrosis, the EMT hallmarks including epithelial cell markers (E-cadherin and CK-18) and mesenchymal cell markers (\u0026alpha;-SMA and VIM) were determined using qRT-PCR (Fig. 1A), Western blot (Fig. 1B) and immunohistochemistry (IHC) (Fig. 1C) techniques in both IUA tissues and normal tissues. The results, as depicted in Fig. 1a-1c, demonstrated a reduction of the levels of E-cadherin and CK-18 whereas the levels of \u0026alpha;-SMA and VIM were obviously enhanced in IUA tissues compared to normal tissues, suggesting involvement of EMT in IUA progression. To identify the circRNAs expression profiles involved in IUA progression, a circRNA microarray analysis on 3 paired IUA samples and adjacent normal samples under hysteroscopy was performed. The criteria for selecting differentially expressed circRNAs was absolute fold change \u0026gt; 2.0 and p value \u0026nbsp; less than 0.05. Among them, 348 differentially expressed circRNAs were reported in circBase (www.circbase.org/) and 235 differentially expressed circRNAs were detected in all samples including 72 upregulated circRNAs and 163 downregulated circRNAs. A heatmap was constructed to show different expressed circRNAs (Fig. 1D). Furthermore, top 10 circRNAs were selected for further verification by qRT-PCR by using designed divergent primers and found that only hsa_circ_0132015 and hsa_circ_0079474 were confirmed. We further found that hsa_circ_0079474 had more significant upregulation in IUA tissues and more abundance in IUA and normal tissues than hsa_circ_0132015 which was selected for further analysis (Fig. 1E). Furthermore, the expressions of E-cadherin and CK-18 were decreased while \u0026alpha;-SMA and VIM were increased in TGF-\u0026beta;1-induced EECs than control EECs using qRT-PCR (Fig. 1F), Western blot (Fig. 1G) and immunocytochemistry (ICC) (Fig. 1H). The expression of hsa_circ_0079474 was also higher in TGF-\u0026beta;1-induced EECs than control EECs (Fig. 1I).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacteristics of hsa_circ_0079474\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnalysis of circBase showed that hsa_circ_0079474 is originated from back splicing of exons 6-13 of human gene DGKB (also known as hsa_circ_DGKB-009) which was located on chromosome 7, with the length 812 nt (Fig. 2A). To confirm the results of the expression of hsa_circ_0079474 in IUA tissues, Sanger sequencing on the PCR products of hsa_circ_0079474 was performed. Sanger sequencing verified the back-splicing junction with the expected size and predicted splicing site in the PCR products (Fig. 2B). Agarose gel electrophoresis\u0026nbsp;of reverse transcribed RNA (cDNA) and genomic DNA (gDNA) were performed by convergent and divergent primers for hsa_circ_\u0026shy;0079474. The results demonstrated that the divergent primers could amplify products from cDNA but not gDNA (Fig. 2C). As a confirmation of the stability of hsa_circ_0079474, the total RNA samples were treated with RNase R. The results of qRT-PCR displayed that the levels of linear DGKB (DGKB mRNA) was decreased significantly under RNase R treatment, whereas circDGKB-009 was resistant to the digestion of RNase R (Fig. 2D). The stability of hsa_circ_0079474 was further confirmed by treating EECs with Actinomycin D (a transcription inhibitor) for different periods of time. Results disclosed that the linear DGKB mRNA level decreased gradually with time but the level of\u0026nbsp;circDGKB-009\u0026nbsp;was more stable and resistant to\u0026nbsp;Actinomycin D\u0026nbsp;administration (Fig. 2E). In addition,\u0026nbsp;hsa_circ_0079474 was mainly located in cytoplasm measured by\u0026nbsp;cytoplasmic and nuclear fractionation assay, with the positive control U6 in nucleus and GAPDH in cytoplasm, respectively (Fig. 2F). In brief, these findings indicated that\u0026nbsp;hsa_circ_0079474\u0026nbsp;is a circRNA which is\u0026nbsp;localized in the cytoplasm, has good stability, and may involve in the occurrence and development of IUA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHsa_circ_0079474 promoted the progression of EMT\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the functional roles of\u0026nbsp;hsa_circ_0079474, gain-of-function and loss-of-function assays were performed, respectively. Firstly, overexpression plasmid of\u0026nbsp;hsa_circ_0079474 was constructed and transfected to Ishikawa cells. The level of hsa_circ_0079474 was increased more than 60,000-fold in hsa_circ_0079474-overexpression of Ishikawa cells compared with control by qRT-PCR (Fig. 3A). Secondly, three specific siRNAs targeting hsa_circ_0079474 were transfected into Ishikawa cells to silence hsa_circ_0079474 expression. The results demonstrated that si-hsa_circ_0079474-3 had the highest efficiency and was selected for further functional trials (Fig. 3B).\u003c/p\u003e\n\u003cp\u003eSubsequently, CCK-8\u0026nbsp;assays and Edu staining assays were conducted to determine the impact of hsa_circ_0079474 on the proliferation of Ishikawa cells.\u0026nbsp;The findings disclosed that hsa_circ_0079474\u0026nbsp;overexpression led to improvement of the proliferation of Ishikawa cells whereas silence of hsa_circ_0079474 suppressed the proliferation of Ishikawa cells (Fig. 3C, D). Flow cytometry analysis elucidated that the up-regulation of hsa_circ_0079474\u0026nbsp;resulted in a decrease in the proportion of the G0/G1 phase but an increase in the percentage of the S phase of the cell cycle in Ishikawa cells. Conversely, down-regulation of hsa_circ_0079474 resulted in elevation in the percentage of the G0/G1 phase and a reduction in the proportion of the S phase of the cell cycle in Ishikawa cells, demonstrating that hsa_circ_0079474-overexpressing promoted cell cycle progression while down-regulation of hsa_circ_0079474 led to cell cycle arrest (Fig. 3E). Nevertheless, both up-regulation and down-regulation of hsa_circ_0079474 were found to have no significant impact on the proportion of G2/M phase of the cell cycle in Ishikawa cells. To further investigate the effects of hsa_circ_0079474 on EMT, qRT-PCR, Western blot and ICC were employed. The obtained results validated that overexpression of hsa_circ_0079474 led to a reduction in the levels of epithelial cell markers E-cadherin and CK-18 while increasing the levels of mesenchymal cell markers\u0026nbsp;\u0026alpha;-SMA and VIM. Conversely, knockdown of\u0026nbsp;hsa_circ_0079474 yielded the opposite outcomes (Fig. 3F-H).\u0026nbsp;In conclusion,\u0026nbsp;hsa_circ_0079474 showed effects of promotion of cell proliferation, acceleration of cell cycle progression and improvement of EMT.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHsa_circ_0079474\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eserved as a sponge for miR-630\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt was postulated that hsa_circ_0079474 exerts its function through the mechanism of miRNA sponges, which is a prevalent mechanism for circRNAs. Circular RNA Interactome and CircBank datasets were employed to predict the potential binding miRNAs of hsa_circ_0079474. As exhibited in Venn diagram in Fig. 4A, three candidate miRNAs (hsa\u0026shy;-miR-630, hsa\u0026shy;-miR-892a and hsa\u0026shy;-miR-935) were identified to have putative binding sites on hsa_circ_0079474. The miRanda dataset was performed to suggest that miR-630, miR-892a and miR-935 could potentially bind to hsa_circ_0079474, with binding scores of 162, 156 and 151, and a total free energy of -14.39\u0026nbsp;kCal/Mol,\u0026nbsp;-14.32\u0026nbsp;kCal/Mol and\u0026nbsp;-16.67 kCal/Mol, respectively. These findings indicate that miR-630 is the most likely to bind hsa_circ_0079474. The expression levels of the aforementioned three miRNAs were subsequently assessed in both IUA and\u0026nbsp;normal\u0026nbsp;tissues. \u0026nbsp;It was observed that miR-630 exhibited a significant decrease in IUA tissues compared to\u0026nbsp;normal\u0026nbsp;tissues whereas no significant differences were found in miR-892a and miR-935\u0026nbsp;(Fig. 4B). Additionally, the levels of miR-630 were determined in Ishikawa cells with overexpression of hsa_circ_0079474. The results indicated a significant decrease in miR-630\u0026nbsp;expression (Fig. 4C). Consequently, miR-630 was selected for further experimentation.\u0026nbsp;The predicted binding sites of hsa_circ_0079474 to miR-630 were also displayed (Fig. 4D) and dual-luciferase reporter assay was performed to\u0026nbsp;confirm the\u0026nbsp;interaction between\u0026nbsp;hsa_circ_0079474 and miR-630. The wild-type hsa_circ_0079474 sequences, which included potential miR-630 binding sites, as well as a mutation version, were inserted into the psiCHECK2 luciferase reporter. Results manifested that miR-630 remarkably inhibited the luciferase activity while miR-630 inhibitor notably enhanced luciferase activity in hsa_circ_0079474-wt reporter while not in the hsa_circ_0079474-mut reporter. This suggested that hsa_circ_0079474 has the ability to bind to miR-630 (Fig. 4E). Additionally, to understand the molecular mechanisms of hsa_circ_0079474 in IUA progression, subcellular localization was conducted in EECs by FISH. As presented in Fig. 4F. FISH results exhibited co-localization of miR-630 and hsa_circ_0079474 in the cytoplasm of EECs. RIP assays demonstrated that hsa_circ_0079474 was remarkably immunoprecipitated by Ago2 antibody in miR-630-transfected EECs compared to miR-NC-transfected EECs (Fig. 4G). These results confirmed that hsa_circ_0079474 served as a sponge for miR-630 in EECs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMiR-630 was downregulated in IUA and alleviated EMT process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further explore the involvement of miR-630 during IUA development, EECs\u0026nbsp;and\u0026nbsp;TGF-\u0026beta;1-induced EECs\u0026nbsp;were collected and subjected to qRT-PCR to evaluate the expression of miR-630. Results manifested that the expression of miR-630 were lower in both IUA tissues and TGF-\u0026beta;1-induced EECs compared to normal tissues and EECs, respectively. \u0026nbsp;(Fig. 5A).\u0026nbsp;In addition, transfection of miR-630 mimics and miR-630 inhibitors\u0026nbsp;into EECs confirmed the upregulation and downregulation of miR-630 (n=3) (Fig. 5B). Subsequently, CCK-8 and Edu staining assays were employed to observe the impacts of miR-630 on the property of proliferation of EECs. Results revealed that miR-630 inhibited the proliferation of EECs whereas suppression of miR-630 promoted the proliferation of EECs (Fig. 5C, D). Flow cytometry analysis further exhibited that miR-630 was found to have a restraining effect on the cell cycle while inhibition of miR-630 induced cell cycle progression (Fig. 5E). The results of expression of EMT markers proved that miR-630 enhanced the levels of epithelial cell markers (E-cadherin and CK-18) while decreasing the expression of mesenchymal cell markers (\u0026alpha;-SMA and VIM)\u0026nbsp;(Fig. 5F-H).\u0026nbsp;In summary,\u0026nbsp;miR-630 yielded properties of inhibition of cell proliferation, cell cycle progression and EMT.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMiR-630 could directly target YAP1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the potential target genes of miR-630, various datasets including miRDB, miRWalk, miRTarbase, RNACentral and TargetScan were utilized to predict. As a result, only one candidate gene (YAP1) was filtrated for further analysis (Fig. 6A). The two predictive binding sites on YAP1-3\u0026rsquo;UTR for miR-630 by miRanda were presented in Fig. 6B. To validate the binding between miR-630 and YAP1, dual-luciferase report assays was further conducted. Wild-type (Wt) YAP1-3\u0026rsquo;UTR and miR-630 binding site Mut luciferase reporter plasmids were transfected along with miR-630 mimic and miR-630 inhibitor. Results indicated that the relative luciferase activity of wt YAP1-3\u0026rsquo;UTR group was marked declined when transfected miR-630 mimic but remarkably increased when transfected miR-630 inhibitor. Conversely, no significantly differences of the relative luciferase activity were observed in mut YAP1-3\u0026rsquo;UTR groups (Fig. 6C). In addition, the expression of YAP1 was evaluated using qRT-PCR, Western blot and ICC following transfection with miR-630 mimic and miR-630 inhibitor and indicated that elevation of miR-630 dramatically inhibited the expression of YAP1 whereas downregulation of miR-630 distinctly improved the expression of YAP1 (Fig. 6D-F). These results confirmed that miR-630 could target YAP1 directly.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYAP1 was increased in IUA and promoted EMT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the underlying role of YAP1 in IUA development, samples of IUA tissues, normal tissues, EECs and\u0026nbsp;TGF-\u0026beta;1-induced EECs\u0026nbsp;were collected and subjected to qRT-PCR, Western blot and IHC/ICC analysis. Results concluded that the level of YAP1 in IUA tissues and\u0026nbsp;TGF-\u0026beta;1-induced EECs\u0026nbsp;was significantly higher than adjacent\u0026nbsp;normal\u0026nbsp;tissues and EECs respectively (Fig. 7A-C). To further explore the role of YAP1 in IUA development, YAP1-overexpression plasmid and siRNAs were transfected into EECs. The efficiency of transfection was confirmed using qRT-PCR. The transfection of YAP1-overexpression plasmid into EECs resulted in an increased level of YAP compared to the control group. (Fig. 7D). Next, three specific siRNAs targeting YAP1 were designed and transfected into EECs to silence YAP1 expression. It was illustrated that si-YAP1-3 exhibited the highest efficiency and was selected for further experiments (Fig. 7E).\u0026nbsp;Further functional trials demonstrated that YAP1 overexpression resulted in a dramatically improvement of cell proliferation, cell cycle progression and EMT while knockdown of YAP1 disclosed opposite effects (Fig. 7F-K).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHsa_circ_0079474 promotes IUA progression via miR-630/YAP1 axis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurthermore, our study proposed to investigate the involvement of miR-630 in the regulation of hsa_circ_0079474 in Ishikawa cells. It was observed that the presence of hsa_circ_0079474 significantly enhanced cell proliferation, facilitated cell cycle progression, and promoted EMT. However, these effects were effectively counteracted by the restoration of miR-630 expression (Fig. 8A-F). Conversely, the depletion of hsa_circ_0079474 in Ishikawa cells was rescued by the administration of miR-630 inhibitor (Fig. 8G-L). The findings elucidated that hsa_circ_0079474 exerted its effects on Ishikawa cells, at least in part, by acting as a sponge for miR-630.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHsa_circ_0079474 promoted IUA via miR-630 \u003cem\u003ein vivo\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression of E-cadherin and CK-18 were decreased while the expression of \u0026alpha;-SMA and VIM increased compared with sham group by qRT-PCR and immunohistochemical staining, confirming that EMT was involved in the progression of IUA in rat (Fig.9A, B). Masson staining was applied to determine the degree of fibrosis. In IUA group, more fibrotic area was found than that of control group, suggesting that the IUA rat model was established successfully (Fig. 9B).\u0026nbsp;It was further validated that IUA+AAV-hsa_circ_0079474 group notably decreased levels of E-cadherin and CK-18, accompanied by increased levels of \u0026alpha;-SMA and VIM, as well as a greater presence of collagen fibers compared to the IUA group by qRT-PCR, IHC and Masson staining\u0026nbsp;(Fig.9c-9d). Conversely, injection of si-hsa_circ_0079474 yielded contrasting results of AAV- hsa_circ_0079474 (Fig. 9C, D).\u0026nbsp;In IUA+miR-630 agomir group, intrauterine injection of miR-630 agomir demonstrated higher levels of E-cadherin and CK-18 whereas lower levels of \u0026alpha;-SMA and VIM, as well as lower rate of fibrotic area, which disclosed contrasting effects of\u0026nbsp;hsa_circ_0079474\u0026nbsp;using qRT-PCR, IHC and Masson staining\u0026nbsp;(Fig. 9E-F). Furthermore, administration of miR-630 antagomir exhibited opposite effects\u0026nbsp;(Fig. 9E-F). However, these effects of\u0026nbsp;AAV- hsa_circ_0079474 were at least partly reversed by administration of miR-630 agomir (Fig. 9G, H). Furthermore, the effects of hsa_circ_0079474-siRNA-chol were reversed at least partially by miR-630 antagomir (Fig. 9G, H). These findings displayed that\u0026nbsp;hsa_circ_0079474 improved IUA via miR-630 \u003cem\u003ein vivo\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn recent years, emerging evidences have confirmed the roles of circRNAs in the development of multiple diseases, especially tumors. However, only three circRNAs have been reported thus far to be implicated in the progression of IUA. CircPlekha7 was first reported circRNA which played anti-fibrotic role in the IUA [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. CircPTP4A2 contributed to endometrial repair progression by targeting miR-330-5p/PDK2 axis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. CircPTPN12 accelerated IUA progression by regulating miR-21-5 p/∆Np63α pathway [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In our study, we screened a new circRNA, hsa_circ_0079474, which was derived from the exons 6\u0026ndash;13 of DGKB (also named circDGKB-009) by circRNA expression profiles. Although another circDGKB, hsa_circ_0133622 (circDGKB-017) was reported in the progression of neuroblastoma [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], the function of hsa_circ_0079474 has not been reported yet. We further confirmed the circular characteristics of hsa_circ_0079474 and revealed the mechanism of hsa_circ_0079474 in IUA development.\u003c/p\u003e \u003cp\u003eIt is proposed that the ceRNA mechanism for circRNAs, which means circRNAs might act as sponges of miRNAs to prevent miRNAs-triggered inhibition on the expression of target genes. In our present study, we investigated whether hsa_circ_0079474 exerted pro-fibrotic role in IUA by the above circRNA-miRNA-mRNA pathway. We confirmed that hsa_circ_0079474 was mainly located in the cytoplasm by FISH. Further bioinformatic analysis, luciferase reporter, and RIP assays validated that miR-630 was the target of hsa_circ_0079474 and was degraded by hsa_circ_0079474, as well as YAP1 was the target gene of miR-630.\u003c/p\u003e \u003cp\u003eMiR-630 is a miRNA which encoded by MIR630 gene (NC_000015.10) on 15q24.1[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. MiR-630 had been associated with multiple neoplastic and non-neoplastic conditions. The impacts of miR-630 on human malignancies had been fully elucidated but the results were controversial. MiR-630 represented anti-tumor properties in non-small cell lung cancer [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], esophageal cancer [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], thyroid cancer [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], cervical cancer [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and breast cancer [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. On the other hand, miR-630 acted as an oncogenic miRNA in colorectal cancer [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], ovarian cancer [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], renal cell cancer [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], prostate cancer [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and acute lymphoblastic leukemia [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. MiR-630 also had been involved in non-tumor disease such as IgA nephropathy [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and cataract [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, further studies were needed to investigate the potential role of miR-630 in fibrosis. In our experiment we examined the expression of miR-630 in IUA tissues and TGF-β1-induced EECs and proved that miR-630 yielded inhibition of IUA, contributing to a deeper understanding of its functional role.\u003c/p\u003e \u003cp\u003eSeveral target genes of miR-630 had been validated by dual-luciferase reporter assay. For example, miR-630 could directly target vimentin, thus suppressing non-small cell lung cancer [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. MiR-630 targeted IGF1R to regulate response to HER-targeting drugs and cancer progression in HER2 overexpressing breast cancer [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. MiR-630 inhibited EMT by targeting Slug in hepatocellular carcinoma [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. YAP1 had been previously confirmed as a target gene of miR-630 [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. YAP1 had been involved in the progression of fibrosis including pulmonary fibrosis [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], cardiac fibrosis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and liver fibrosis [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In this experiment, we validated the binding of miR-630 and YAP1 by dual-luciferase reporter assay again and elucidated the effect of hsa_circ_0079474 on IUA via miR-630/YAP1 axis.\u003c/p\u003e \u003cp\u003eIn conclusion, hsa_circ_0079474 sponges miR-630 to facilitate YAP1 expression, thus improving EMT in IUA (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e10\u003c/span\u003e). This finding provided a new insight into the molecular mechanism of circRNA in IUA and revealed that targeting hsa_circ_0079474/miR-630/YAP1 might be a promising therapeutic alternative for IUA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTissues collection\u003c/h2\u003e \u003cp\u003e This study was authorized by the Ethics Committee of the First Affiliated Hospital with Nanjing Medical University (approval number 2022-SRFA-321). Paired fibrotic endometrial samples and adjacent normal endometrial samples were sampled from IUA patients who received hysteroscope at the First Affiliated Hospital with Nanjing Medical University. Informed consent was provided by all participants. We instantly frozen all samples in liquid nitrogen after hysteroscopy, and then stored them at -80\u0026deg;C until they were needed. For endometrial tissue collection, endometrial tissue biopsies were obtained from women with regular menstrual cycles and no exposure to steroids at least 3 months who received hysterectomy or subtotal hysterectomy for leiomyoma by curettage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCircRNA expression profiles\u003c/h2\u003e \u003cp\u003eTotally three paired IUA samples and adjacent normal samples were selected for circRNA expression profiles. The Agilent Human ceRNA Microarray 2019 was applicated in our study and further data analysis of all samples were performed by OE Biotechnology (Shanghai, China). Quantification of total RNA was performed using the NanoDrop ND-2000 (Thermo Scientific) and RNA integrity was evaluated by Agilent Bioanalyzer 2100 (Agilent Technologies). As directed by the manufacturer, samples were labeled, microarray hybridized, and washed in accordance with their instructions. Differentially expressed circRNAs were identified through fold change and \u003cem\u003eP\u003c/em\u003e value calculated using the t-test. The threshold was a fold change\u0026thinsp;\u0026ge;\u0026thinsp;2.0 and a \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and cell lines\u003c/h2\u003e \u003cp\u003eFor primary endometrial epithelial cells (EECs) culture, endometria were instantly placed in culture medium and isolation began within 2 hours of collection. Endometria were cut up and digested in 1mg/ml collagenase I (Beijing Solarbio Science \u0026amp; Technology Co., Ltd) at 37\u0026deg;C for 60min. Following centrifugation, samples were filtered with a 150 nm strainer and a 38 nm strainer, respectively. The samples retained on the 38 nm strainer were regarded as EECs. The EECs were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium/F12 (DMEM/F12) medium (Sigma-Aldrich). To induce EMT, TGF-β1(100ng/ml, Bioss) was added in the medium for EECs. Ishikawa cells and HEK-293T cells (Procell, Wuhan, China) were routinely cultured in DMEM medium with high glucose (KeyGEN, Nanjing, China). Both media contained 10% fetal bovine serum (Procell, Wuhan, China), 100U/ml penicillin and 0.1mg/ml streptomycin (Beyotime, Shanghai, China). Incubation was performed at 37\u0026deg;C in 5% CO2 and 95% air in a humidified atmosphere.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time polymerase chain reaction (qRT-PCR)\u003c/h2\u003e \u003cp\u003eAn extraction of total RNA was carried out using Trizol reagent (Beyotime, Shanghai, China) from tissue samples and cells. The Nanodrop 2000 spectrophotometer (Thermo Fisher, USA) was used to measure the concentration and purity of the RNA samples. The cDNA was synthesized by HiScript III RT SuperMix for qPCR (+\u0026thinsp;gDNA wiper) (R323-01, Vazyme, Nanjing, China) for circRNAs and mRNAs. MiRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (MR101-01, Vazyme, Nanjing, China) was used for cDNA synthesis of miRNAs. The quantitative PCR was conducted by ChamQ SYBR qPCR Master Mix and miRNA Universal SYBR qPCR Master Mix (Q311-02 and MQ101-01, Vazyme, Nanjing, China) according the manufacturer\u0026rsquo;s instructions on an ABI 7500 RealTime PCR System (Applied Biosystems, USA). The level of miRNAs was normalized by U6, while circRNAs and mRNAs by glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The calculation of RNA expression was determined using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method. The primers were synthesized by Tsingke Biotechnology Co., Ltd (Beijing, China) and listed in Additional file 1: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eConfirmation of circRNA\u003c/h2\u003e \u003cp\u003eTo verify the circular characteristic of hsa_circ_0079474, the linear and circular transcripts of DGKB were amplified using convergent and divergent primers in both complementary DNA (cDNA) and genomic DNA (gDNA) isolated from IUA and adjacent normal samples. Agarose gel electrophoresis was applicated to separate the PCR products. Sanger sequencing was performed to confirm the sequence and back-splicing sites of hsa_circ_0079474.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eActinomycin D and RNase R treatment\u003c/h2\u003e \u003cp\u003eTo inhibit transcription, 2\u0026micro;g/ml Actinomycin D and dimethylsulfoxide (negative control) were added into the culture medium. For RNase R treatment, total RNA was subjected to 2U/\u0026micro;g RNase R (Geneseed, Guangzhou, China) and incubated at 37℃ for 20min. Following treatment, the RNAs were then subjected to reverse transcription using linear and circular DGKB primers for qRT-PCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of cytoplasmic and nuclear RNA\u003c/h2\u003e \u003cp\u003e Cytoplasmic and nuclear fractionation assay was carried out by Nuclear and Cytoplasmic Extraction Kit (Beyotime, Shanghai, China) and Trizol reagent (Beyotime, Shanghai, China) in tissue samples following the guidelines provided by the manufacturer. Isolated cytoplasmic and nuclear RNA were then reverse transcribed by qRT-PCR to detect the relative expression of linear and circular DGKB.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of plasmid and cell transfection\u003c/h2\u003e \u003cp\u003eThe sequences of hsa_circ_0079474 and YAP1 were amplified and subsequently inserted into pLC5-ciR vector (Geneseed, Guangzhou, China) and pENTER vector (Vigene, Ji\u0026rsquo;nan, China) to construct hsa_circ_0079474 or YAP1-overexpressing plasmid, respectively. Three small interfering RNAs (siRNAs) targeting the junction sequence of hsa_circ_0079474 (si-hsa_circ_0079474-1, forward 5\u0026rsquo;-UACUGGUCUGAGAAUGAAUTT-3\u0026rsquo; and reverse 5\u0026rsquo;-AUUCAUUCUCAGACCAGUATT \u0026minus;\u0026thinsp;3\u0026rsquo;; si-hsa_circ_0079474-2, forward 5\u0026rsquo;-AGUGGUACUGGUCUGAGAATT-3\u0026rsquo; and reverse 5\u0026rsquo;-UUCUCAGACCAGUACCACUTT-3\u0026rsquo;; si-hsa_circ_0079474-3, forward 5\u0026rsquo;- GGUACUGGUCUGAGAAUGATT-3\u0026rsquo; and reverse 5\u0026rsquo;-UCAUUCUCAGACCAGUACCTT-3\u0026rsquo;), YAP1 (si-YAP1-1, forward 5\u0026rsquo;-GGUGAUACUAUCAACCAAATT-3\u0026rsquo; and reverse 5\u0026rsquo;-UUUGGUUGAUAGUAUCACCTT-3\u0026rsquo;; si-YAP1-2, forward 5\u0026rsquo;- CAUUAACGACUAGAUUAAATT-3\u0026rsquo; and reverse 5\u0026rsquo;- UUUAAUCUAGUCGUUAAUGTT-3\u0026rsquo;; si-YAP1-3 forward 5\u0026rsquo;- GCCACAGAUUAAGAUUAUATT-3\u0026rsquo; and reverse 5\u0026rsquo;- UAUAAUCUUAAUCUGUGGCTT-3\u0026rsquo;) and control non-specific oligonucleotides (si-NC, forward 5\u0026rsquo;- UUCUCCGAACGUGUCACGUTT-3\u0026rsquo; and reverse 5\u0026rsquo;- ACGUGACACGUUCGGAGAATT-3\u0026rsquo;) were obtained from Vigene (Ji\u0026rsquo;nan, China). MiR-630 mimics, miR-630 inhibitor and scrambled control (miR-NC) were synthesized by Sangon (Shanghai, China). Above siRNAs and miRNAs were transfected into Ishikawa cells by application of Lipo8000\u0026trade; Transfection Reagent (Beyotime, Shanghai, China) according to the manual of manufacturer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eCell counting kit-8 (CCK-8) assay\u003c/h2\u003e \u003cp\u003eCCK-8 assay was conducted to evaluate the proliferation of Ishikawa cells. Cells were seeded and cultured in 96-well plates for 0, 24h, 48h, 72h and 96h. CCK-8 solution (New Cell \u0026amp; Molecular Biotech, Suzhou, China) was added to each well (10\u0026micro;l/plates). The absorbance at 450 nm was measured by a spectrophotometer (Thermo Scientific) after 1 h of incubation at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e5-Ethynyl-2\u003csup\u003e\u0026rsquo;\u003c/sup\u003e-Deoxyuridine (EdU) assay\u003c/h2\u003e \u003cp\u003eThe EdU assay was conducted with a EdU Cell Proliferation Kit (Beyotime, Shanghai, China). The Ishikawa cells were fixed in 4% paraformaldehyde after being incubated for 2 hours with 10\u0026micro;M EdU. Hoechst-33,342 (Beyotime, Shanghai, China) was applicated to stain the nucleus. An EdU-positive cell percentage was calculated from images acquired with a fluorescence microscope (Nikon, Japan).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eCell cycle assay\u003c/h2\u003e \u003cp\u003eTransfected Ishikawa cells were fixed in 70% ethanol at 4\u0026deg;C overnight and stained with propidium iodide (Beyotime, Shanghai, China). Cell cycle analysis was performed by flow cytometer (BD FACSCanto, USA). The ratio of cells in different phase was counted and compared.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eDual-luciferase reporter assay\u003c/h2\u003e \u003cp\u003eTo validate the binding of hsa_circ_0079474 and miR-630, as well as miR-630 and YAP1, dual-luciferase reporter assay was conducted. Wild type (WT) and mutant (MUT) sequence of hsa_circ_0079474 and YAP1 of predicted binding sites with miR-630 were synthesized and cloned into psiCHECK2 reporter vector. The reporter vector, miR-630 mimics, miR-630 inhibitor and negative control were transfected in HEK-293T cells by Lipofectamine 2000 (Invitrogen, USA). Activity of firefly and renilla luciferase were evaluated by \u003cem\u003eTransDetect\u003c/em\u003e\u0026reg; Double-Luciferase Reporter Assay Kit (TransGen, Beijing, China) in accordance to the manufacture\u0026rsquo;s manual.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eFluorescence in situ hybridization (FISH)\u003c/h2\u003e \u003cp\u003eHybridization was conducted overnight at 37\u0026deg;C with specific probes with fluorescence label of hsa_circ_0079474 and miR-630. 4',6-diamidino-2-phenylindole (DAPI) was counterstained for 20 min to stain nucleus. The sections were observed under a fluorescence microscope (Nikon, Japan). Nuclei stained blue while hsa_circ_0079474 which was labeled with Cy3 stained red, and miR-630 which was labeled with fluorescein isothiocyanate (FITC) stained green. FISH probes were sequenced as follows.: hsa_circ_0079474, 5\u0026prime;-tattcattctcagaccagtaccact-3\u0026prime;; miR-630, 5\u0026prime;- AGUAUUCUGUACCAGGGAAGGU-3\u0026prime;; Both the probes were synthesized by Geneseed (Guangzhou, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eBioinformatic analysis\u003c/h2\u003e \u003cp\u003eThe binding between hsa_circ_0079474 and miR-630 was predicted by Circular RNA Interactome (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://circinteractome.nia.nih.gov/\u003c/span\u003e\u003cspan address=\"https://circinteractome.nia.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and CircBank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.circbank.cn/\u003c/span\u003e\u003cspan address=\"http://www.circbank.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) while the interaction between miR-630 and YAP1 was predicted by TargetScan (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.targetscan.org/vert_71/\u003c/span\u003e\u003cspan address=\"https://www.targetscan.org/vert_71/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), miRTarbase (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://mirtarbase.cuhk.edu.cn/php/index.php\u003c/span\u003e\u003cspan address=\"https://mirtarbase.cuhk.edu.cn/php/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), miRCentral (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://rnacentral.org/\u003c/span\u003e\u003cspan address=\"https://rnacentral.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), miRDB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mirdb.org/\u003c/span\u003e\u003cspan address=\"http://mirdb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and miRWalk (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mirwalk.umm.uni-heidelberg.de/\u003c/span\u003e\u003cspan address=\"http://mirwalk.umm.uni-heidelberg.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eRNA immunoprecipitation (RIP) assay\u003c/h2\u003e \u003cp\u003eThe RIP assay was performed by RIP Kit (Geneseed, Guangzhou, China) in accordance to the instructions of the manufacturer. Briefly, Ishikawa cells were collected, lysed and Ago2 antibodies (normal control) or IgG (Ishikawa cells) were conjugated with magnetic beads and incubated overnight at 4\u0026deg;C. After washing the beads, proteinase K was used to dissolve proteins. An agarose gel electrophoresis and quantitative RT-PCR were conducted after RNA extraction.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eTotal proteins from cells and tissues were extracted by RIPA Lysis Buffer (Beyotime, Shanghai, China) and the concentration of protein were performed by BCA protein assay kit (Bioss, Beijing, China). Proteins were separated and resolved in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred into nitrocellulose membranes and then subjected to blocking of skim milk and incubation of primary antibodies of E-cadherin (AF6759, Beyotime, Shanghai, China), CK-18 (CSB-MA000253, CUSABIO, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cusabio.com/\u003c/span\u003e\u003cspan address=\"https://www.cusabio.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), α-SMA (ab5694, Abcam, UK), Vimentin (ab8978, Abcam, UK), YAP1(bs-3605R, Bioss, Beijing, China), GAPDH (AF0006, Beyotime, Shanghai, China) and horseradish peroxidase-conjugated secondary goat anti-mouse/rabbit antibody (Beyotime, Shanghai, China).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnimal experiments\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor establishment of IUA model, 8-week-old female Sprague-Dawley rats (Animal Core Facility of Nanjing Medical University) were utilized. Rats were housed under a 12h:12h light-dark cycle with free access to water and food. The protocols of the experiment were conducted according to the guidelines of Institutional Animal Care and Use of Laboratory Animal and authorized by the Laboratory Animal Ethics Committee of Nanjing Medical University (approved number: IACUC-1912051). The rat model of IUA was conducted by endometrial scraping in accordance to our method which previous reported [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Briefly, rats were anesthetized by intraperitoneal injection of pentobarbital (40 mg/kg). Rats were divided into eight groups (n\u0026thinsp;=\u0026thinsp;3 per group): sham-operation group, IUA group, IUA\u0026thinsp;+\u0026thinsp;adeno-associated virus (AAV)-hsa_circ_0079474 group, IUA\u0026thinsp;+\u0026thinsp;si-79474 group, IUA\u0026thinsp;+\u0026thinsp;miR-630 agomir group, IUA\u0026thinsp;+\u0026thinsp;miR-630 antagomir group, IUA\u0026thinsp;+\u0026thinsp;AAV-hsa_circ_0079474\u0026thinsp;+\u0026thinsp;miR-630 agomir group and IUA\u0026thinsp;+\u0026thinsp;si-79474\u0026thinsp;+\u0026thinsp;miR-630 antagomir group. For sham-operation group, the abdomen was cut and sutured without treating the uterus. For other groups, after the Y‑type uterus was exposed, the bilateral uterus was cut and scraped by a razor blade. The wound was then sutured for IUA group. Intrauterine injection of AAV-hsa_circ_0079474 (OBiO Technology Corp., Ltd, Shanghai, China) (1*10^10 viral genomes per rat), miR-630 agomir (5nmol per rat), miR-630 antagomir (10nmol per rat) and hsa_circ_0079474-siRNA-chol (10nmol per rat) were utilized in the left uterus of rats while AAV-vector, agomir-NC (5nmol per rat) antagomir-NC (10nmol per rat) and siRNA-NC (10nmol per rat) were injected in the right uterus of the corresponding rat. Rats were sacrificed by intraperitoneal injection of pentobarbital (100 mg/kg,) and uteri were cut and collected 14 days after surgery. For histological evaluation, tissues were fixed in 4% paraformaldehyde, embedded in paraffin for cutting slices. Masson staining was utilized to determine the degree of fibrosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical staining\u003c/h2\u003e \u003cp\u003eFor immunohistochemical (IHC) staining, paraffin sections were firstly deparaffinized and rehydrated. For immunocytochemistry (ICC), samples were first fixed in formalin for 20min. Sections/Samples were then immersed into 0.3% Triton X-100. Antigen retrieval was performed with 0.1 mmol/L sodium citrate at 95\u0026ndash;100\u0026deg;C for 15 minutes, followed by incubation in 3% hydrogen peroxide to block endogenous peroxidase activity. Primary antibodies (listed in the Western blotting) were incubated overnight at 4\u0026deg;C then incubated with secondary antibody. Sections were then stained with 3,3\u0026rsquo;-diaminobenzidine (ZhongShan Golden Bridge, Beijing, China) for visualization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation and analyzed by SPSS 24.0. Comparisons between two groups were performed by Student\u0026rsquo;s t test. while three or more groups were conducted by one-way analysis of variance (ANOVA) followed by Dunnett\u0026rsquo;s test. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were regarded as statistical differences while \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 were considered as significant statistical differences.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFig.10 was drawn by Figdraw. Thank you for pathologists of the First Affiliated Hospital with Nanjing Medical University (Yang Li and Guoqiang Ping) for preparing tissue sections.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Young Scholars Fostering Fund of the First Affiliated Hospital of Nanjing Medical University (PY2021003).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWestendorp IC, Ankum WM, Mol BW, Vonk J. 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Exp Ther Med. 2021;22(3):968. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Intrauterine adhesion, fibrosis, hsa_circ_0079474, miR-630, YAP1","lastPublishedDoi":"10.21203/rs.3.rs-3767908/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3767908/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInsufficient understanding exists of the molecular mechanisms underlying circRNA involvement in IUA\u003cstrong\u003e \u003c/strong\u003eand requires further investigation. This research aims to examine the role of hsa_circ_0079474 (circDGKB-009) and its potential mechanisms in intrauterine adhesion (IUA). A circRNA microarray was utilized to identify differences in circRNA expression between fibrotic endometrial samples and normal endometrial samples. Subsequent studies confirmed the expression and biological functions of hsa_circ_0079474 both in vivo and in vitro using various experimental techniques such as CCK-8, EdU, flow cytometry, FISH, RT-PCR, Western blot and IHC/ICC. The interactions between hsa_circ_0079474 and miR-630, as well as miR-630 and YAP1 were determined using dual-luciferase reporter assay and RNA immunoprecipitation. Hsa_circ_0079474 was dramatically elevated in IUA tissues compared to normal tissues. Hsa_circ_0079474 was found to enhance cell proliferation, expedite cell cycle progression, and facilitate epithelial-mesenchymal transition (EMT). Mechanistically, hsa_circ_0079474 acted as a sponge for miR-630, resulting in upregulation of YAP1 expression. This, in turn, promoted the progression of IUA. Hsa_circ_0079474 improves IUA by regulating the miR-630/YAP1 axis, providing a novel understanding of the molecular mechanisms underlying circRNA in IUA.\u003c/p\u003e","manuscriptTitle":"Hsa_circ_0079474 facilitates epithelial-mesenchymal transition in intrauterine adhesion via miR-630/YAP1 axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-25 15:33:51","doi":"10.21203/rs.3.rs-3767908/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c8c52c16-ed8f-44fc-8caa-ecdac0ae444e","owner":[],"postedDate":"January 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":27906857,"name":"Health sciences/Diseases/Reproductive disorders"},{"id":27906858,"name":"Health sciences/Biomarkers/Diagnostic markers"}],"tags":[],"updatedAt":"2024-01-25T15:33:51+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-25 15:33:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3767908","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3767908","identity":"rs-3767908","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}