Circ_0007331 knock‐down suppresses the progression of endometriosis via miR‐200c‐3p/HiF‐1α axis

Abstract Endometriosis is considered a benign gynaecological disease with cancer‐like characterizations, which has a high incidence among women of reproductive age. However, this disease has so far lacked timely diagnosis and effective treatment owing to its unclear aetiology. In this study, we identified aberrant high expression of circ_0007331 in ectopic endometrial cells by comparing the endometrial samples from patients with and without endometriosis. Further functional experiments revealed that circ_0007331 knock‐down effectively suppressed the viability, proliferation and invasive capacity of ectopic endometrial cells. Additionally, we attempted to define the molecular mechanism of circ_0007331 in the initiation and progression of endometriosis. Circ_0007331 acted as a miRNA sponge for miR‐200c‐3p to indirectly regulate the function of HIF‐1α, which plays a key role in the local angiogenesis and hypoxic mechanisms of ectopic endometrium. A final in vivo experiment confirmed that circ_0007331 knock‐down could suppress the development of endometriosis through down‐regulating the expression of HIF‐1α. Collectively, we preliminarily characterized the role and possible insights of circ_0007331/miR‐200c‐3p/HIF‐1α axis in the proliferation and invasion of ectopic endometrial cells. We hope that by exploring the potential function and molecular mechanism of circ_0007331, we can increase our biological insight into the pathogenesis of endometriosis, which will bring the new ways for the diagnosis and therapy of this disease.

theories including Sampson's theory of retrograde menstruation have been proposed, the pathogenesis of this disease is not thoroughly understood. 4 Besides, the clinical diagnosis of endometriosis is also extremely rough and hysteretic. Due to the lack of adequately sensitive and specific signs as well as blood tests, the current gold standard for the diagnosis of endometriosis mainly consists of invasive laparoscopic surgery and subsequent histopathological examination, which will cause a delay in disease confirmation and bring great pain to patients. 5 Compared with traditional surgical diagnosis, non-invasive or minimally invasive biomarkers not only help to shorten the diagnosis delay and improve patient compliance, but also have the advantages of low risk and low cost. Therefore, the World Endometriosis Society (WES) and the World Endometriosis Research Foundation (WERF) have identified the development of new non-invasive biomarkers as scientific issues that urgently need to be solved in this disease field. 6 Circular RNAs (circRNAs) are a type of regulatory RNAs that lack canonical 5′ cap and 3′ poly-A tail attributed to their covalent closedloop structures. 7 As the competitive endogenous RNAs, circRNAs regulate the expression of multiple genes at the transcriptional and post-transcriptional levels by sponging downstream microRNA (miRNA). Most meaningfully, circRNAs have been increasingly used as the biomarker in the diagnosis, therapy and prognosis of various diseases due to its advantages of abundant expression level, high stability and covalent terminals. 8 Circ _0081001 is identified as a potential biomarker for the diagnosis of osteosarcoma, which can dynamically monitor and reflect the disease development of the patients. 9 Hsa_circ_0000291 is regarded as a promising therapeutic target for gastric cancer (GC) due to its effective inhibitory role on the metastasis and growth of GC cells by being silenced or down-regulated. 10 Circ_0079593 predicts an adverse prognosis owing to its strong association with the cell growth and invasion of glioma. 11 The research of circRNAs in endometriosis has also made preliminary progress. Analysis of clinical samples by microarray and bioinformatics methods revealed 88 differentially expressed circRNAs between eutopic and normal endometrium, of which 11 were up-regulated and 77 were down-regulated. 12 In addition, circ_103470 and circ_101102 were found to regulate epithelial-mesenchymal transition (EMT) of endometriosis through sponge miR-141-5p. 13 Further functional analysis showed that downstream mRNAs of these differentially expressed circRNAs are mainly involved in immune-inflammatory response and cell cycle regulation. Obviously, the potential of circRNAs as a biomarker to monitor the initiation and progression of diseases is also applicable to endometriosis.
Although a series of differentially expressed circRNAs have been widely discovered in various diseases, little is known about their biogenesis processes and underlying mechanisms. It has been learned that the regulatory function of circRNAs mainly depends on its directly bound downstream miRNAs. 14 MicroRNAs (miRNAs) are a type of conservative small regulatory non-coding RNAs that can suppress the expression of target genes. As miRNAs are involved in key biological processes relied on cell life such as cell proliferation, apoptosis, development and differentiation, they are considered as vital regulators of gene expression. Circ_0000285 is reported to interact with miR-654-3p to act as a miRNA sponge which in turn activates MAPK6 in diabetic nephropathy, thereby causing podocyte damage. 15 MiR-25-3p could affect the unchecked proliferation of endometrium in endometriosis by directly regulating specificity protein 1 (Sp1). 16 Recently, a report delving into the molecular basis of the circRNA/miRNA network in the pathogenesis of endometriosis found that hsa_circ_0067301/miR-141-5p/Notch-1 axis promotes the EMT process of this disease. 17 In this study, we identified aberrant high expression of circ_0007331 in endometriosis by comparing endometrial tissue samples from patients with endometriosis and normal individuals.
Further in vivo and in vitro experiments demonstrated the presence of the circ_0007331/miR-200c-3p/HIF-1α axis in the positive regulation of ectopic endometrial cell viability, proliferation and invasion. We hope that by exploring the potential function and molecular mechanism of circ_0007331, we can increase our biological insight into the pathogenesis of endometriosis, which will bring the new ways for the diagnosis and therapy of this disease.

| MATERIAL S AND ME THODS
Ethical approvals were obtained from the Ethics Committee of the Renmin Hospital of Wuhan University. All collected samples were obtained subsequent to receive full written informed consent from the patients. Relevant animal experiments were conducted complied with the institutional ethical guidelines.

| Clinical samples collection
All samples were taken from female patients aged 35-45 with a regular menstrual cycle (25-32 days). The collection time was the proliferative phase of the menstrual cycle, and patients did not receive any hormonotherapy for at least 6 months pre-operatively.

| Cell culture and identification
The experimental cell lines in this study were divided into normal endometrial (NE) cells and eutopic endometrial (EE) cells, which derived from the 0.5-1 mm thick part of the endometrial functional layer of patients without endometriosis and patients with ovarian chocolate cysts, respectively. Immediately after the samples were gathered, they were cultured in sterile saline containing 1.5% antibiotics and antifungal. Afterwards, these tissues were washed, minced, digested with collagenase and trypsin and filtered with a 50-µm-diameter nylon mesh to obtain the desired cells. Cells were grown in Dulbecco's modified Eagle's medium/Nutrient Mixture F-12 Ham (DMEM/F12, Invitrogen) replenished with 10% foetal bovine serum (FBS, Sigma-Aldrich) in a humidified incubator containing 5% CO2 at 37°C.

| Cell transfection
The knock-down treatments are required for evaluating the functions of circ_0007331 in EE cells. Therefore, the cor-

| Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
The mRNA expressions of circ_0007331, miR-200c-3p and HIF-1α in the experimental groups were examined by qRT-PCR. In strict accordance with the manufacturer's guidelines, total RNAs were

| Western blot analysis
All HIF-1α proteins were extracted from the endometrial cells lysed in RIPA lysis buffer (RIPA, Beyotime), and the protein concentrations were evaluated by bicinchoninic acid (BCA) analysis (Beyotime). The same amounts of protein were separated using 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore). Then, the membranes were blocked using 5% skim milk powder at room temperature for 1 hour, followed by incubation with primary antibody against HIF-1α (Catalogue#3716, Cell Signaling technology) at 4°C overnight. The next day, membranes were then conjunct with secondary antibodies (1:5000) at room temperature for 1 hour. Finally, every protein band was visualized by an enhanced chemiluminescence kit (Pierce Biotechnology; Thermo Fisher Scientific, Inc) and related data were quantified by Image Lab Software.

| Cell counting kit-8 (CCK-8) assay
The viability of EE transfected cells was detected by the CCK-8 method. Cells were inoculated into 96-wells with a density of 5000 cells per well and incubated until cell attachment. Then, these seeded cells were cultured for 1, 2, 3, 4 and 5 days separately after transfection. Following treatment, 10 µL of the CCK-8 solution (KeyGen) was supplied to every well. Finally, a microplate spectrophotometer (BioTek) was employed to detect the OD value of each well at 450 nM.

| EdU assay
The effects on the proliferation of EE cells were determined by EdU assay using the BeyoClick™ EdU-555 Kit (Beyotime Biotechnology) in line with the protocols of manufacturer. Transfected EE cells were seeded in 96 wells and cultured for 2 days. After fixed with 4% paraformaldehyde, cells were added with 5-ethynyl-2'-deoxyuridine (EdU, 10 µM each) and cultivated at 37°C for 2 hours. These cells were subsequently stained using Apollo Dye Solution and DAPI.
Finally, the EdU-positive cells were visualized and analysed through the fluorescence microscope (Olympus).

| Transwell invasion assay
Cell invasion ability was analysed by transwell assay using BioCoat

Matrigel invasion chambers (BD Biosciences). Transwell chambers
were settled in a sterile 24-well cell culture plate initially. The EE transfected cells were added to FBS-free DMEM to prepare cell resuspension with a density of 2.5 × 105 cells/mL. 100 µL of the above cell suspension added onto the top chamber and 600 µL of culture medium containing 10% FBS were seeded to the under chamber, respectively. Then, these treated cell culture plates were incubated with 5% CO2 at 37°C for 36 hours. Afterwards, the cells that migrated to the lower chamber were fixed with 70% MeOH and stained with 0.5% crystal violet in turn. Finally, the number of migrated cells was determined by microscopic counting.

| Immunohistochemistry
After fixed in 4% paraformaldehyde, embedded in paraffin and sliced successively, the mouse model samples were incubated with the corresponding primary antibodies against HIF-1α (Catalogue#3716, Cell Signaling technology) at 4°C for 24 hours. The intensity and percentage of staining were appraised by H-score (a semi-quantitative grading system). The images were captured and analysed using an optic microscope (Olympus).

| Statistical analysis
The results of each experiment were repeated three times, and the experimental data were represented as means ± standard deviation (SD). The analyses were mainly interpreted applying with the software statistical SPSS version 19.0 (IBM). The statistical variances between the control group and experimental group were calculated through the unpaired two-tailed t test. Other statistical variances among multigroups were assessed by utilizing one-way ANOVA or two-way ANOVA. A P value of less than .05 (P < .05) was determined as statistically significant.

| The expression of circ_0007331 is abnormally elevated in endometriosis
To preliminarily explore the possible relevance between circ_0007331 and endometriosis, we detected the mRNA levels of

| Circ_0007331 knock-down results in decreased cell proliferation and invasion of the EE cells in endometriosis
We

| Circ_0007331 knock-down suppresses the proliferation of EE cells by down-regulating the expression of HIF-1α
Given that the strong connection between HIF-1α and the adhesion as well as the proliferation of endometrial cells in endometriosis, we

| MiR-200c-3p is predicted to mediate the regulation of HIF-1α by circ_0007331
CircRNAs are known to regulate the downstream gene through sponging the corresponding miRNAs. We therefore next

| Circ_0007331 affects the proliferation and invasion of EE cells by sponging miR-200c-3p to regulate HIF-1α
Likewise, we also evaluated the changes of miR-200c-3p in en-

| D ISCUSS I ON
Although endometriosis has been identified as a debilitating disease with benign characteristics, the various symptoms that accompany it seriously disturb the quality of life of thousands of women of reproductive age. Due to the complexity of endometriosis symptoms associated with various diseases, there is no clear consensus on the pathological mechanism of this disease at the moment.
Endometriosis is firstly defined as an inflammatory disease, characterized by the activation of macrophage migration inhibitory factor (MIF), tumour necrosis factor-alpha (TNF-α) and multiple interleukins (IL-6, IL-8, IIL-1β) and the up-regulation of inflammatory biomarkers including monocyte chemotactic protein 1 (MCP-1) and C-reactive protein (CRP). 18 Additionally, endometriosis also exhibits cancer-like characterizations such as neovascularization, cell invasion and unrestrained cell proliferation. 19 The disease is not regarded as a malignant disease, while researchers have recognized the possibility of endometriotic tissues transforming into malignant ovarian cancer.
Accurate detection and early diagnosis are crucial for the therapy of endometriosis, but owing to the diversity and concealment of its inchoate symptoms, the current gold standard for diagnosis still consists of invasive surgery and the following histopathology examination, which often results in 5-10 years delay in the management of this disease. Therefore, the research in this field mainly focuses on the development of novel non-invasive biomarkers from the biological fluids of patients for the early detection of diseases recently. The reported potential biomarkers for endometriosis usually originate from blood and urine, including miRNAs, lncRNAs, growth factors, glycoproteins and proteins related to immunity or angiogenesis. The representative glycoprotein served as a biomarker for endometriosis is CA-125, which usually characterizes the degree of severity in this disease. 20 The single levels of inflammatory cytokines IL-1, IL-6 and IL-8 do not reflect the pathological development of endometriosis, but combined usage has the potential to mark diseases. 21 The expression level of insulin-like growth factor-1 (IGF-1) has been reported to be significantly different in stages I-II-IV and I-II of endometriosis, but subsequent studies have added much controversy for its use as a biomarker. 22 TC0101441 was found to exhibit a statistically significant positive correlation with infertility and chronic pelvic pain, which has the potential to diagnose the severity of the disease. 23 Although the above-mentioned biomolecules can more or less reflect the single-dimensional physiological characteristics of endometriosis, due to the lack of specificity and sensitivity for this pathology, there is still a certain gap from clinical application. In recent years, miRNA has been predicted as an ideal biomarker for the diagnosis of endometriosis due to its advantages of high humoral stability and high tissue specificity. However, it has been found that no single miRNA could provide sufficient specificity and sensitivity to this disease after several further studies.
Mounting evidence showed that circRNAs can be adopted to assess the diagnosis and prognosis of various diseases and even as a therapeutic target, such as cancer, CNS disease, cardiovascular disease and certain endocrine dysfunction. 24 However, there is little research on the biological function and molecular mechanism of circRNAs in endometriosis. Circ_0004712 and circ_0002198 are the earliest published novel biomarkers promising for the diagnosis of endometriosis. 12 CircRNA_103237 was proved to suppress cell proliferation and invasion in inchoate secretory endometrium of women with endometriosis through sponge miR-34. 25 EMT, as a prerequisite for endometriosis, was found to be regulated by the signalling pathway hsa_circ_0067301/miR-141/Notch. 17 In this study, HIF-1α is a transcription factor present in human and mammalian cells under hypoxic conditions, which plays a vital role in mediating angiogenesis and promoting cell proliferation. 29 Overexpression of HIF-1α has been found in a variety of cancers, including pancreatic cancer, breast cancer, prostate cancer, colon cancer and lung cancer. 30 Previous studies showed that the occurrence and development of endometriosis were closely related to local angiogenesis and hypoxic mechanisms. Further quantitative analysis proved that HIF-1α protein levels in ectopic endometriosis tissues were higher than eutopic endometrial tissues. 31 Growing evidence shows that HIF-1α plays a key role in the development of ectopic endometrium by mediating genes related to oestrogen production, angiogenesis, proliferation and inflammation. 32 Moreover, HIF-1α inhibitors were gradually adopted in the therapeutic strategy for endometriosis like rapamycin, 2-methoxyestradiol and HDAC inhibitors. However, previous research mainly focused on the functional study of HIF-1α in endometriosis. Even if the mechanism was involved, the key point was often on the downstream signals rather than the upstream regulatory factors of HIF-1α such as circRNAs or miRNAs. We reported the first cir-cRNAs that affects the regulation of HIF-1α on endometriosis and explored its possible mechanism. This not only provides a potential tool for other researchers to continue to explore the deeper mechanism of HIF-1α in endometriosis, but also offers a choice for the therapeutic strategy of endometriosis targeting HIF-1α, which is different from small molecule inhibitors.
However, our research remains a key question. The initial aim of this work was to search for a non-invasive biomarker for the diagnosis of endometriosis. Unfortunately, we only evaluated and investigated the differential distribution and biological function of circ_0007331 in ectopic tissues. Although circ_0007331 has considerable potential as a therapeutic target, it needs further studies in peripheral blood or menstrual blood for the clinical applications as a non-invasive diagnostic biomarker.
In summary, our results show that circ_0007331 is abnormally highly expressed in patients with endometriosis. Both in vitro or in vivo, circ_0007331 knock-down suppresses the progression of endometriosis by inhibiting the proliferation and invasion of ectopic endometrial cells. Further mechanism studies confirmed that the molecular basis of this function is the circ_0007331/miR-200c-3p/ HIF-1α axis. Our work not only reveals the potential therapeutic targets for future drug interventions in endometriosis, but also provides novel insights for the fine regulation of HIF-1α in endometriosis.

CO N FLI C T O F I NTE R E S T
No potential conflict of interest was reported by the authors.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon reasonable request.