ALKBH5 promotes autophagy and progression by mediating m6A methylation of lncRNA UBOX5-AS1 in endometriosis

Long noncoding RNA (lncRNA) and N6-methyladenosine (m6A) methylation modification have recently been suggested as potential functional modulators in ovarian endometriosis, however, the function and mechanism of m6A-modified lncRNA in ovarian endometriosis remain poorly understood. In this study, we demonstrated that lncRNA UBOX5-AS1 expression was significantly elevated in ovarian endometriosis tissue and primary ectopic endometrial stromal cells. The expression of lncRNA UBOX5-AS1, which has m6A modifications, was highly positively correlated with demethylase Alk B homologous protein 5 (ALKBH5) expression and autophagy. Functional studies revealed that increased ALKBH5 and lncRNA UBOX5-AS1 expression promoted cell autophagy, proliferation, and invasion in endometriosis in vitro. LncRNA UBOX5-AS1 mediates ALKBH5-regulated autophagy, proliferation, and invasion. ALKBH5-mediated autophagy facilitates cell proliferation, migration, and invasion. In vivo, the knockdown of ALKBH5 inhibited endometriotic lesion growth. Mechanistically, we observed that ALKBH5 mediated the m6A demethylation of lncRNA UBOX5-AS1 and promoted its expression. Thus, our findings highlight that ALKBH5/lncRNA UBOX5-AS1 might serve as potential targets for ovarian endometriosis therapy in the future. NEW & NOTEWORTHY In the present study, we investigated the role and potential molecular mechanism of long noncoding RNA (lncRNA) UBOX5-AS1 in ovarian endometriosis progression. Combined with the aforementioned, we proposed the hypothesis that lncRNA UBOX5-AS1 regulated by Alk B homologous protein 5 (ALKBH5)-mediated N6-methyladenosine (m6A) modification contributes to the progression of ovarian endometriosis progression.

Endometriosis (EMs) is a prevalent disease among women of childbearing age, with an incidence rate of 10%–15%, and has been steadily increasing over the years (1, 2). EMs presents a complex clinical challenge, marked by a high risk of recurrence, making it a leading cause of infertility and chronic pelvic pain. This condition imposes significant psychological and physical burdens on women of childbearing age (3, 4). To date, a definitive understanding of the pathogenesis of endometriosis remains elusive because its molecular mechanisms remain largely unclarified. Thus, it is necessary to further elucidate the mechanisms of endometriosis progression to develop novel therapeutic targets. Autophagy is a highly conserved biological process in eukaryotic cells that involves the use of lysosomes to remove damaged organelles and foreign bodies, thereby preserving cellular homeostasis and enhancing resilience under adverse conditions (5, 6). Under stress conditions such as hypoxia and starvation, cells activate the Unc-51-like kinase (ULK) complex, initiating the phosphorylation of Beclin1 and the formation of phagophores. Autophagosomes, guided by autophagy-associated genes (ATGs), are subsequently transported to lysosomes and merge, where they form autolysosomes that degrade their contents, contributing to cell survival (7). Abnormal autophagy activation is implicated in various pathological processes, including inflammation (8), tumor invasion (9), and aging-related diseases (10). Although endometriosis is classified as a benign disease, it exhibits aberrant biological behavior reminiscent of malignant tumors, such as rapid growth, aggressive invasion, extensive proliferation, and angiogenesis. Our previous research has confirmed the abnormal activation of autophagy in the ectopic endometrium of patients with EMs (11–13). Autophagy plays a dual role in the context of endometriosis: it promotes cell survival by reducing susceptibility to apoptosis while also facilitating the invasion and migration of ectopic endometrial cells through diverse mechanisms. Nonetheless, the precise causative factors behind the abnormal activation of autophagy in endometriosis warrant further investigation. In recent years, long noncoding RNAs (lncRNAs) have emerged as pivotal regulators of cellular biology because of their ability to modulate gene expression and influence various cellular functions, including autophagy, which is often mediated by RNA binding proteins (14, 15). Our earlier studies revealed abnormal upregulation of lncRNA UBOX5-AS1 in the ectopic lesions of patients with endometriosis, where it is implicated in fostering invasion and epithelial to mesenchymal transition of endometrial epithelial cells (16). However, although autophagy is significant in disease development, the underlying mechanisms by which lncRNA UBOX5-AS1 operates in the regulation of autophagy in ovarian endometriosis have yet to be identified. N6-methyladenine (m6A) modification is recognized as one of the most common and abundant forms of chemical RNA modification in eukaryotes (17, 18). Our previous research revealed that the overall level of m6A in the ectopic endometrium of patients with EMs is lower than that in the normal endometrium, with a concurrent significant increase in the expression of the classical m6A demethylase Alk B homologous protein 5 (ALKBH5) (19, 20). ALKBH5, among the m6A demethylases, is known to be involved in a spectrum of biological activities, including invasion (21), migration (22), and proliferation (23). Studies have demonstrated that ALKBH5 can influence the stability and expression of lncRNAs, consequently regulating downstream biological events (24, 25). In addition, ALKBH5 can modulate the process of autophagy in various cell types, including myocardial, testicular, and pulmonary cells, through alterations in m6A modifications (26–28). However, the underlying mechanisms through which ALKBH5 is involved in lncRNA UBOX5-AS1-induced autophagy have not been fully elucidated. In the present study, we investigated the role and potential molecular mechanism of lncRNA UBOX5-AS1 in ovarian endometriosis progression. We proposed the hypothesis that lncRNA UBOX5-AS1 regulated by ALKBH5-mediated m6A modification contributes to the progression of ovarian endometriosis progression.

Patients and Specimens All the samples were obtained from the Department of Obstetrics and Gynecology, affiliated with the Union Hospital of Huazhong University of Science and Technology. Thirty-four samples of normal endometrium (NE) were obtained from individuals with tubal infertility undergoing diagnostic curettage. Ectopic endometrium (EC) samples were obtained from 32 individuals with stage III-IV ovarian endometriosis, diagnosed through ultrasonography and pathology. All patients included had regular menstrual cycles and had not received hormonal therapy for at least 6 mo before participation. All samples were collected during the proliferative stage of the menstrual cycle, which was determined by the date of the last menstrual period and histological criteria. Informed consent forms were signed by all patients. The use of clinical samples was approved by the Ethics Committee of Tongji Medical College at Huazhong University of Science and Technology (IORG No.: IORG0003571). The clinical data of the patients are summarized in Supplemental Table S1. Isolation and Culture of Primary Endometrial Stromal Cells Primary normal endometrial stromal cells (NESCs) were isolated from eight cases of normal endometrium from women with tubal infertility but without endometriosis (EMs). Primary ectopic endometrial stromal cells (EESCs) were isolated from eight cases of ectopic endometrium from patients with ovarian endometriosis. The collected fresh tissue was rinsed with PBS. The tissue was subsequently minced using scissors and incubated with preheated 0.1% type II collagenase (Sigma-Aldrich, St. Louis, MO). The mixture was then incubated in a shaker at 37°C for 45 min. The mixture was sequentially filtered through aseptic sieves with 150-μm and 38-μm pore sizes to remove epithelial cells and undigested tissues. The mixture was then centrifuged at 1,000 rpm for 5 min. Red blood cell lysis buffer was added, the mixture was mixed, and the cells were further centrifuged to isolate primary endometrial stromal cells. Both NESCs and EESCs were cultured in DMEM/F12 with 20% FBS and incubated in a 5% CO2 atmosphere at 37°C. Cells up to the third passage were used for experiments. Immunofluorescence was used to assess the purity of the isolated endometrial stromal cells (ESCs) by assessing the expression of the epithelial marker E-cadherin (Abcam, ab40772, 1:50) and the mesenchymal marker vimentin (Abcam, ab92547, 1:50) (Supplemental Fig. S1). Characterization and Identification of Isolated Primary Human Endometrial Stromal Cells Immunocytochemistry assays were conducted to detect the mesenchymal marker vimentin and the epithelial marker E-cadherin. In brief, primary human endometrial stromal cells were isolated and plated into a six-well plate at a density of 20,000 cells/well. The cells were grown until they reached ∼60% confluence. To fix the cells, they were incubated with 4% paraformaldehyde at 4°C for 15 min. The cells were subsequently permeabilized with 0.3% Triton X-100 for 10 min to enhance antibody penetration. To prevent nonspecific binding of the antibodies, the cells were blocked with 1% bovine serum albumin (BSA) in PBS for 1 h at room temperature. The cells were then incubated overnight at 4°C with primary antibodies against E-cadherin (1:50; Abcam, Cambridge, UK) and vimentin (1:50; Abcam, Cambridge, UK). Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:500; Servicebio Biotech, Wuhan, PR China) were then applied for 1 h at 37°C. After the cells were washed with PBS, Mayer’s hematoxylin was used for nuclear counterstaining. The stained cells were observed and photographed using an Eclipse TE2000-S microscope system (Nikon UK Ltd., Surrey) and Image-Pro Plus software (Media Cybernetics UK, Berkshire). Cell Culture and Transfection Assay Human endometrial stromal cells (HESCs; ATCC, VA) were cultured in DMEM/F12 medium (Gibco, CA) supplemented with 10% fetal bovine serum (Gibco, CA) under conditions of 5% CO2 at 37°C. The design and construction of the ALKBH5 overexpression plasmid, the LncUBOX5-AS1 overexpression plasmid, and the control plasmids were performed by DesignGene (Shanghai, PR China). Corresponding negative control plasmids were also constructed. Small interfering RNAs (siRNAs) used to suppress ALKBH5, and their corresponding negative control siRNAs, were synthesized by RiboBio (Guangzhou, PR China). Transfection experiments were conducted using Lipofectamine 3000 (Invitrogen, CA), following the manufacturer’s instructions. The efficiency of cell transfection was observed under a fluorescence microscope, and subsequent experiments were performed after confirming the transfection efficiency. Detailed sequence information is listed in the Supplemental Material. Protein Extraction and Western Blotting Analysis Total protein from tissues and cells was extracted using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, PR China) containing PMSF (Sigma-Aldrich, St. Louis, MO). The protein was denatured by heating at 95°C for 10 min and then stored at −80°C. Equal amounts of protein (30 μg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated protein was transferred to polyvinyl difluoride membranes (PVDF, Millipore, MA) and incubated in 5% skim milk in Tris-buffered saline with 0.05% Tween-20 (TBST) at room temperature for 1 h to block nonspecific binding. The membranes were then incubated with the appropriate primary antibody overnight at 4°C. The next day, after washing with TBST three times, the membranes were incubated with a goat anti-rabbit HRP secondary antibody (1:400, Proteintech, Wuhan, PR China) at room temperature for 1 h. After three additional washes with TBST, the membranes were incubated with enhanced chemiluminescence (ECL) solution, and the results were observed using an imaging system. ImageJ software was used for quantification. The characteristics of the antibodies used for the Western blotting assay are listed in Supplemental Table S3. Total RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction Analysis The expression of lncRNA UBOX5-AS1 was assessed using quantitative real-time polymerase chain reaction (qRT-PCR) in normal endometrium (n = 26), ectopic endometrium from patients with ovarian endometriosis (n = 24), NESCs (n = 26), and EESCs (n = 24). The cDNA primer sequences were designed based on the cDNA sequence and synthesized by QingKe Biotechnology Co., Ltd. (Shanghai, PR China). Total RNA extraction was conducted using TRIzol reagent (Tsingke Biotech Co. Ltd, Xi’an, PR China). The RNA concentration was determined using a Nanodrop 2000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). The purity of the RNA was assessed by measuring the A260/A280 ratio, with a range of 1.8–2.0 considered acceptable. Reverse transcription of the total RNA was performed using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, PR China). For qRT-PCR analysis of RNA expression, Tsingke Master qPCR Mix (SYBR Green I with UDG, Xi'an, PR China) was used. GAPDH served as the reference gene for normalization. The data were analyzed using the 2−ΔΔCT method. The specific primer sequences are provided in Supplemental Table S2. Immunohistochemical Staining Analysis Fresh surgical samples were initially fixed in 10% formaldehyde for 24 h before being embedded in paraffin blocks. The formalin-fixed, paraffin-embedded endometrial tissues were subsequently sectioned at a thickness of 5 µm and mounted on alcohol-cleaned glass slides. The sections were dewaxed in xylene, followed by rehydration through a graded alcohol series to water. Antigen retrieval was achieved by heating the sections in citrate buffer (pH 6.0). To quench endogenous nonspecific peroxidase activity, the sections were incubated in a 50% ethanol solution containing 3% H2O2 for 30 min. The sections were subsequently blocked with a protein block for 30 min and then with bovine serum albumin for another 30 min. They were then incubated overnight at 4°C with primary antibodies against ALKBH5 (1:100, Proteintech, 16837-1-AP), ATG12 (1:100, Proteintech, 11122-1-AP), and LC3 (1:100, CST, No. 2775). After being washed with PBS, the sections were incubated for 30 min at room temperature with peroxidase-labeled anti-rabbit IgG (1:500; Wuhan Boster Biotechnology Co. Ltd., Wuhan, PR China, BA1039). Finally, the slides were treated with diaminobenzidine (DAB) substrate (Beyotime, Shanghai, PR China), counterstained with hematoxylin, dehydrated, and mounted. The optical density of the representative images was analyzed using IPP software (Image-Pro Plus 6.0) following the immunohistochemical (IHC) analysis. The characteristics of the antibodies used for immunohistochemistry are listed in Supplemental Table S2. Transmission Electron Microscopy To study autophagosomes at the ultrastructural level, HESCs were cultured for 24 h under either hypoxic or normoxic conditions. After treatment, the cells were washed three times with PBS and incubated with trypsin for 2 min. The cells were then collected by centrifugation at 1,000 g for 5 min. The cell pellets were suspended in 2.5% glutaraldehyde in 0.1 M Na-phosphate buffer (pH 7.4) and fixed overnight at 4°C. Postfixation was performed using 1% OsO4 in 0.1 M cacodylate buffer (pH 7.4) for 3 h, followed by two washes in 0.1 M Na-phosphate buffer for 15 min each. The cells were then dehydrated at 25°C using a graded ethanol series and gradually infiltrated with an epoxy resin mixture (812 resin embedding kit). Sequential polymerization of the samples was performed at 37°C for 12 h, 45°C for another 12 h, and finally 60°C for 24 h. Ultrathin sections of 50–70 nm was cut using an LKB microtome and mounted on single-slot copper grids. The sections were subjected to double staining with uranyl acetate and lead citrate and were examined with a transmission electron microscope (TEM; FEI Tecnai G20, Super Twin, Double Tilt, LaB6 Gun). GFP-mRFP-LC3 Adenoviral Vector The GFP-mRFP-LC3 adenoviral vector, constructed by HanBio Technology Co., Ltd. (HanBio, Shanghai, PR China), was used to study autophagy in HESCs. The cells were cultured on slides placed in six-well plates and allowed to reach a confluence of 50%–70% before infection. Following the manufacturer’s instructions, adenovirus (multiplicity of infection, MOI = 100) was added directly to the culture medium and incubated for 24 h. After incubation, the cells were washed with precooled PBS and fixed at room temperature with 4% formaldehyde for 15 min. The formaldehyde was then removed by rinsing with PBS, and the slides were sealed with glycerin. Autophagy was visualized and photographed using a confocal laser scanning microscope (Olympus America Inc., Center Valley, PA). The presence of green fluorescent protein (GFP) and monomeric fluorescent protein (mRFP) puncta was quantified to assess autophagic flux. Scratch Wound Healing Assay Human endometrial stromal cells were seeded into six-well plates. After transfection, the cells were ensured to reach 90%–95% confluence. Using a marker pen, at least three straight lines were marked on the underside of each well. A 200-μL pipette tip was subsequently used to create a linear scratch perpendicular to the marked lines. The cells were then washed with PBS to remove the detached cells, and medium without FBS was added for continued incubation in a 5% CO2 incubator at 37°C. Cell migration was observed using an inverted microscope (Olympus 600 auto-biochemical analyzer, Tokyo, Japan) at 0 h and 48 h. The calculation for the wound healing distance (%) was as follows: Wound healing distance (%) = (Scratch distance at 0 h − Scratch distance at 48 h)/Scratch distance at 0 h. Transwell Migration/Invasion Assays Transwell migration and invasion assays were conducted in vitro using transwell chambers (Corning Costar, MA) with 8-μm pore membrane filters. For invasion assays, Matrigel (Corning Costar, MA) was used to coat the transwell chambers. Approximately 1 × 105/mL cells were seeded into the upper chamber with serum-free medium, allowing them to migrate or invade the lower chamber, which contained culture medium supplemented with 20% fetal bovine serum. After 24 h of incubation, noninvasive cells remaining on the upper surface were removed using a cotton swab. The migrated or invaded cells on the underside of the membrane were then fixed with 4% paraformaldehyde and stained with 0.1% crystal violet staining solution. To quantify the results, the number of migrated or invaded cells was counted in five random fields under a light microscope (Olympus 600 auto-biochemical analyzer, Tokyo, Japan). The average values of triplicate experiments were reported, with duplicate wells used for each condition in independent experiments. EdU Assay To evaluate cell proliferation, human endometrial stromal cells were first cultured in six-well plates. The cells were then incubated for 2 h with EdU reagent preheated to 37°C, using an EdU proliferation kit (Beyotime, Shanghai, PR China). Following incubation, the culture medium was discarded, and the cells were fixed with 1 mL of 4% formaldehyde at room temperature for 15 min. After fixation, the formaldehyde was removed, and the cells were washed three times for 5 min each with 1 mL of immunostaining blocking solution. Subsequently, 1 mL of immunostaining permeabilization solution was added, and the cells were incubated at room temperature for another 15 min. Once the reaction mixture was prepared, cell proliferation was observed and photographed using a fluorescence microscope. N6-Methyladenosine Modification Prediction To predict potential m6A modification sites within the lncRNA UBOX5-AS1 transcript, we used two reputable online tools: RMBase v2.0 (29) and SRAMP (30). These tools use epitranscriptome sequencing data and machine learning patterns. In addition, we used the m6A2Target database (http://m6a2target.canceromics.org) to identify potential m6A modification enzymes that might influence lncRNA UBOX5-AS1 expression, drawing insights from MeRIP-seq or RNA-seq profiles. RNA Immunoprecipitation RNA immunoprecipitation (RIP) assays were conducted using a RIP Kit (BerSinBio, Guangzhou, PR China). In brief, a total of 2 × 107 cells were collected and lysed with the provided lysis buffer. Following DNA removal from the lysate, protein A/G magnetic beads conjugated with ALKBH5 (Proteintech, 3 µg), ATG12 (CST, 3 µg), or IgG (BerSinBio, 3 µg) were incubated overnight with the lysate. The enriched RNA from the magnetic beads was subsequently collected. Methylated RNA Immunoprecipitation-Quantitative Real-Time PCR The methylated RNA immunoprecipitation (MeRIP) assay was conducted using the MeRIP Kit provided by BerSinBio in Guangzhou, PR China. Briefly, following cell treatment, cellular RNA was extracted as previously described. Ultrasonication was used to fragment the extracted RNA into ∼300-nucleotide segments. The fragmented RNA mixture was subsequently incubated with anti-IgG (BerSinBio, 4 µg) or anti-m6A (BerSinBio, 4 µg) antibodies for 4 h at 4°C. Balanced protein A/G magnetic beads were then added to the mixture to facilitate the enrichment of RNA. The enriched RNA was subsequently extracted, reverse transcribed, and quantitatively analyzed using qRT-PCR. The specific sequences of primers used in this assay are listed in Supplemental Table S2. RNA Fluorescence In Situ Hybridization and RNA Fractionation Assays When the cell culture reached ∼80% confluence, the cells were detached, replated on slides, and allowed to grow to ∼60% confluence. Afterward, the slides were cleaned, and paraformaldehyde was used to fix the cells at room temperature for 15 min. Fluorescent in situ hybridization (FISH) was conducted using the FISH kit from Pinuofei Biotechnology in Wuhan, PR China. An antifluorescence quenching agent was applied to the slides, and the cellular slides were sealed face-down. Images and observations were acquired using a confocal microscope. In this study, a total of 1 × 107 human endometrial stromal cells were collected. To isolate and extract RNA from both the nucleus and cytoplasm, cytoplasmic and nuclear RNA purification kits from Sangon Biotech (Shanghai, PR China) were used, following the manufacturer’s instructions. The extracted RNA was subjected to reverse transcription and quantitative analysis by qRT-PCR, as described previously. Luciferase Reporter Assay Plasmid vectors (PGL3-CMV-LUC-MCS) containing either the wild-type or mutant m6A consensus sequence of LncUBOX5-AS1 were constructed by DesignGene in Shanghai, PR China. Human endometrial stromal cells were transfected with these plasmids, whereas Renilla luciferase reporters served as internal controls. Following a 48-h incubation, luciferase activity was assessed using the Dual-Lucy Assay Kit from Solarbio in Beijing, PR China. Actinomycin D Assay Actinomycin D powder, obtained from Selleck in Shanghai, PR China, was initially dissolved in DMSO at a high concentration of 10 mg/mL. Before use, it was diluted to a concentration of 2 µg/mL for cell treatment. Actinomycin D was introduced 24 h after transfection to inhibit further RNA synthesis at various time intervals (0, 8, 16, and 24 h). Total RNA was subsequently extracted from each group, and the RNA expression levels were quantified using qRT-PCR. The RNA levels at each time point were normalized to those observed at 0 h. Animal Models and Treatment The mouse model of endometriosis was performed, as described previously (31, 32). A total of 39 female C57BL/6 mice, aged 6–8 wk and weighing 18–20 g, were obtained from BIONT Biotechnology Co., Ltd. The mice were randomly assigned to three groups: the donor group (n = 13), the si-ALKBH5 group (n = 13), and the si-Control group (n = 13). The donor mice were anesthetized, and their abdominal cavities were meticulously opened for the surgical procedure. The Y-shaped uterus was ligated and then removed. The excised uterine tissue was cut into small fragments of 2–3 mm and immersed in PBS. The uterine fragments were sutured onto the abdominal walls of recipient mice on both sides, establishing the endometriosis model. To prepare the siRNAs, the siRNA powder was dissolved in aseptic PBS to achieve a concentration of 1 nmol/mL. After a 3-wk recovery period, the mice in the si-ALKBH5 group received intraperitoneal injections of 1 nmol si-ALKBH5 every other day for 3 wk. The control group received intraperitoneal injections of 1 nmol si-Control every 2 days for the same period. At the end of the 3-wk treatment, the mice were euthanized, and the ectopic tissues were collected for analysis. The animal experiments received ethical approval from the Institutional Animal Care and Use Committee (IACUC) at Huazhong University of Science and Technology (IACUC Number: 3396). Statistical Analysis All the data are presented as the means ± standard error (SE). The data were analyzed using GraphPad Prism 8 software (GraphPad Software). For normally distributed data, we used Student’s t test for comparisons between two groups, and ANOVA for multiple group comparisons. For data not normally distributed, we used the Mann–Whitney U test for two-group comparisons and the Kruskal–Wallis test for multiple-group comparisons. Throughout all the figures, we set the level of statistical significance at P < 0.05. Each experiment was performed at least three times.

LncRNA UBOX5-AS1 Is Upregulated in Ovarian Endometriosis Tissues and Primary Ectopic Endometrial Stromal Cells In our initial analysis, we sought to ascertain the potential involvement of lncRNA UBOX5-AS1 in the progression of ovarian endometriosis. We conducted qRT-PCR assay to assess the expression of lncRNA UBOX5-AS1 in normal endometrium and ectopic endometrium from a patient with ovarian endometriosis, primary normal endometrial stromal cells (NESCs) and primary ectopic endometrial stromal cells from a patient with ovarian endometriosis (EESCs). Our findings revealed a significant increase in the expression of lncRNA UBOX5-AS1 in ovarian endometriosis tissues compared with normal endometrial tissues, as illustrated in Fig. 1A. Furthermore, we investigated the expression levels of lncRNA UBOX5-AS1 in primary human endometrial stromal cells. Notably, our results revealed a substantially greater relative abundance of lncRNA UBOX5-AS1 in the EESC group than in the NESC group, as depicted in Fig. 1B. To gain insight into its subcellular localization, we conducted nuclear/cytoplasmic RNA fractionation assays. These experiments confirmed the predominant localization of lncRNA UBOX5-AS1 within the nucleus, as indicated in Fig. 1C. Moreover, our fluorescence in situ hybridization (FISH) analysis further confirmed that lncRNA UBOX5-AS1 predominantly resides within the nucleus of human endometrial stromal cells (HESCs), as depicted in Fig. 1D. In summary, our comprehensive analysis revealed substantial upregulation of lncRNA UBOX5-AS1 in ovarian endometriosis tissues, with primary nuclear localization within HESCs. LncRNA UBOX5-AS1 Promotes Cell Autophagy, Proliferation, and Invasion of HESCs To investigate the biological roles of lncRNA UBOX5-AS1 in the pathological process of ovarian endometriosis, a specific overexpression plasmid for the human lncRNA UBOX5-AS1 (OE-UBOX5-AS1) was used in HESCs. A lncRNA UBOX5-AS1-specific overexpression plasmid that led to significant upregulation of UBOX5-AS1 was selected for further experiments (Fig. 2A). To demonstrate that lncRNA UBOX5-AS1 plays a functional role in autophagy, we assessed autophagic activity in HESCs. Overexpression of lncRNA UBOX5-AS1 significantly increased LC3-II/LC3-I levels and ATG12 expression, suggesting that lncRNA UBOX5-AS1 could promote autophagy (Fig. 2, B and C). In addition, we confirmed the induction of autophagy flux by lncRNA UBOX5-AS1 overexpression in HESCs using the mCherry-GFP-LC3 reporter (Fig. 2, D and E). We also used TEM to evaluate autophagosomes and observed a substantial increase in the accumulation of autophagic vesicles in lncRNA UBOX5-AS1-overexpressing HESCs (Fig. 2, F and G). EdU assays demonstrated that lncRNA UBOX5-AS1 overexpression enhanced the growth capabilities of HESCs (Fig. 2, H and I). Furthermore, wound healing and Transwell assays revealed that lncRNA UBOX5-AS1 overexpression significantly enhanced the migration and invasion capabilities of HESCs (Fig. 2, J–M). Collectively, these findings suggest that lncRNA UBOX5-AS1 is capable of inducing autophagy and promoting the growth, migration, and invasion of HESCs in vitro. ALKBH5 Expression Is Closely Correlated with lncRNA UBOX5-AS1 and Autophagy in Endometriosis Recent advancements in epigenetic research have illuminated the pivotal role of m6A modification at the posttranscriptional level in RNA metabolism. Our curiosity led us to explore whether m6A modification might be linked to the upregulation of lncRNA UBOX5-AS1 in ovarian endometriosis. Our previous study (19) revealed a significant decrease in m6A levels within the ectopic endometrium compared with those in the normal endometrium. To further explore the specific m6A enzyme responsible for regulating lncRNA UBOX5-AS1, we used bioinformatics tools, including m6A2Target, RMBase 2.0, and SRAMP, which leveraged sequencing validation data. RMBase 2.0 data suggested that lncRNA UBOX5-AS1 contains multiple sites of m6A modification (Supplemental Fig. S2A). Our analysis led to the prediction of four highly reliable m6A modification sites and one highly reliable m6A modification site on the lncRNA UBOX5-AS1 transcript (Supplemental Fig. S2B). In addition, our findings highlight several potential writers, erasers, and readers (WERs) of m6A modifications of lncRNA UBOX5-AS1 transcripts, including ELAVL1, FMR1, HNRNPA2B1, IGF2BP1, METTL14, METTL3, YTHDF2, WTAP, RBMX, and ALKBH5 (Supplemental Fig. S3). Our previous study revealed that both the mRNA and protein levels of ALKBH5 were increased in EC compared with NC (19). Therefore, we focused on the role of ALKBH5 in ovarian endometriosis and its regulatory effect on lncRNA UBOX5-AS1. We first examined the expression of ALKBH5 and the autophagy markers ATG12 and LC3 in ectopic and normal endometrial tissues. The protein levels of ALKBH5, ATG12, and LC3-II were significantly greater in the ectopic endometrium (EM) samples than in the normal endometrium (NE) samples (Fig. 3, A and B). We further assessed the protein levels of ALKBH5, ATG12, and LC3-II in primary endometrial stromal cells (ESCs) isolated from EM and NE tissues. Our results demonstrated that the protein levels of ALKBH5, ATG12, and LC3 were markedly greater in EESCs than in NESCs (Fig. 3, C and D). A positive correlation was also observed between ALKBH5 mRNA and lncUBOX5-AS1 levels in both endometriosis tissue and cells (Fig. 3E). Furthermore, the results of the immunohistochemical (IHC) assay confirmed that the ALKBH5, ATG12, and LC3 levels were significantly greater in EMs tissues than in NE tissues (Fig. 3F). Taken together, these data suggest that m6A demethylation is increased and that autophagy is abnormally activated in EMs, potentially contributing to the pathogenesis of endometriosis. ALKBH5-Mediated m6A Modification Enhances lncRNA UBOX5-AS1 Expression To investigate the binding interaction between ALKBH5 and lncRNA UBOX5-AS1 in HESCs, we conducted an RNA immunoprecipitation (RIP) assay. Our findings revealed that lncRNA UBOX5-AS1 was notably enriched in the ALKBH5-bound fraction compared with the control fraction (Fig. 4A). This observation underscores the strong binding affinity between lncRNA UBOX5-AS1 and ALKBH5 in HESCs. Furthermore, our investigation demonstrated that the upregulation of ALKBH5 led to a significant increase in the expression of lncRNA UBOX5-AS1, whereas its knockdown resulted in the opposite effect (Fig. 4, B and C). These results collectively establish that ALKBH5 can bind to lncRNA UBOX5-AS1 and thereby modulate its expression within HESCs. To further confirm that ALKBH5 regulates the stability of lncUBOX5-AS1, we analyzed the stability of lncUBOX5-AS1 in HESCs after treatment with the transcription inhibitor actinomycin D at 5 µg/mL for various durations. As depicted in Fig. 4, D and E, the RNA stability of lncRNA UBOX5-AS1 decreased following the silencing of ALKBH5, whereas the upregulation of ALKBH5 significantly increased the RNA stability of lncRNA UBOX5-AS1. To delve deeper into the requirement for m6A modification of lncRNA UBOX5-AS1 in its regulation by ALKBH5, we conducted luciferase reporter assays. Initially, we predicted potential m6A sites on lncRNA UBOX5-AS1 using an online tool and subsequently constructed wild-type and m6A site mutant dual luciferase plasmids (Fig. 4F). Our observations revealed that luciferase activity was greater in ALKBH5-upregulated HESCs transfected with wild-type lncUBOX5-AS1 than in control HESCs, whereas mutations in the m6A sites abolished this upregulation (Fig. 4G). Furthermore, we found that ALKBH5 knockdown reduced the luciferase activity of the wild-type construct containing lncRNA UBOX5-AS1, whereas mutant lncRNA UBOX5-AS1 was resistant to the effects of ALKBH5 knockdown (Fig. 4H). These results indicate that ALKBH5 regulates lncRNA UBOX5-AS1 expression by modulating its m6A level. To provide further validation, we selected two sites on lncRNA UBOX5-AS1 with the highest confidence and designed primers for methylated RNA immunoprecipitation (MeRIP) assays. MeRIP-PCR assays were performed to measure the m6A levels on lncRNA UBOX5-AS1 after overexpressing or silencing ALKBH5 in HESCs. Notably, the m6A levels of lncRNA UBOX5-AS1 increased significantly following ALKBH5 overexpression, whereas they decreased significantly after ALKBH5 was silenced (Fig. 4, I and J). Collectively, these findings suggest that ALKBH5 serves as a demethylating enzyme for lncUBOX5-AS1, increasing its stability and consequently promoting its expression. ALKBH5 Promotes Cell Autophagy, Proliferation, and Invasion of HESCs To elucidate the functional role of ALKBH5 in autophagy induction and cell biology, we conducted experiments involving the overexpression of ALKBH5 in HESCs (Fig. 5, A and B). We subsequently assessed the levels of autophagy in a series of experiments. The results consistently indicated that ALKBH5 overexpression significantly increased the levels of LC3-II/LC3-I and ATG12 in HESCs (Fig. 5, C and D). Concurrently, the overexpression of ALKBH5 induced autophagy, as evidenced by the notable accumulation of mRFP-GFP-LC3 puncta (Fig. 5, E and F) and an increase in the number of autophagic vesicles (Fig. 5, G and H). These findings collectively demonstrate that ALKBH5 can stimulate the formation of autophagosomes under steady-state conditions. Furthermore, an EdU assay revealed that the proliferation ability of HESCs was augmented following ALKBH5 overexpression in vitro (Fig. 5, I and J). In addition, Transwell and wound healing assays revealed that the upregulation of ALKBH5 significantly enhanced the invasion and migration capabilities of HESCs (Fig. 5, K–N). In conclusion, the evidence suggests that ALKBH5 plays a pivotal role in promoting autophagy while concurrently enhancing the proliferation, invasion, and migration of HESCs. lncRNA UBOX5-AS1 Mediates ALKBH5-Regulated Autophagy, Proliferation, and Invasion of HESCs To elucidate the role of the ALKBH5/UBOX5-AS1 axis in autophagy induction and progression in HESCs, we carried out functional experiments in vitro. Western blotting revealed that genetic inhibition of lncRNA UBOX5-AS1 effectively reversed the ability of ALKBH5 upregulation to promote autophagy in HESCs (Fig. 6, A and B). We further observed that lncRNA UBOX5-AS1 deletion significantly attenuated ALKBH5-induced autophagy, as evidenced by the notable accumulation of mRFP-GFP-LC3 puncta (Fig. 6, C and D) and an increase in the number of autophagic vesicles (Fig. 6, E and F). EdU assays revealed that knockdown of lncRNA UBOX5-AS1 almost fully abolished the proproliferative effect of ALKBH5 on HESCs (Fig. 6, I and J). Wound healing and transwell assays revealed that lncRNA UBOX5-AS1 deletion significantly retarded the ALKBH5-induced invasion and migration capabilities of HESCs (Fig. 6, G, K, H, and L). Collectively, these data indicate that the ALKBH5/lncRNA UBOX5-AS1 axis regulates HESC autophagy activation, growth, and invasion in vitro. ALKBH5 Promotes Proliferation and Invasion via Autophagy in HESCs The induction of autophagy has been reported to contribute to endometrial stromal/epithelial cell migration and invasion (11, 12). Therefore, we examined the effects of the induction/inhibition of autophagy caused by ALKBH5 overexpression/inhibition on HESC proliferation and invasion. We observed that ALKBH5 overexpression significantly increased the proliferation of HESCs, which was consistent with increased migration and invasion (Fig. 7, A–F). Conversely, ALKBH5 suppression with siALKBH5 significantly reduced HESC proliferation, which was consistent with decreased migration and invasion. Moreover, the ALKBH5 overexpression-mediated proproliferative and proinvasive effects were reversed when the cells were cotransfected with the ALKBH5-overexpressing plasmid and the autophagy inhibitor 3-Methyladenine (3-MA) (Fig. 7, A–F). However, the ALKBH5 suppression-mediated antiproliferative and anti-invasive effects were also reversed when the cells were cotreated with ALKBH5 siRNA and the autophagy activator rapamycin (Fig. 7, G–L). Therefore, our results demonstrated that ALKBH5 overexpression promoted HESC proliferation and invasion via autophagy activation. However, ALKBH5 suppression inhibited autophagy, resulting in the inhibition of HESC proliferation and invasion. ALKBH5 Knockdown Blocked Endometriotic Lesion Formation in a Mouse Model of Endometriosis We established an endometriosis (EMs) mouse model by transplanting mouse endometrial tissue fragments into the peritoneal cavity of C57 mouse. To confirm the successful establishment of the EMs mouse model, a C57 mouse was randomly selected and euthanized 2 wk after implantation (Fig. 8A). Single or multiple nodules were clearly observed (Fig. 8B; see animal image). Hematoxylin-eosin (H&E) staining revealed the successful formation of cystic endometrial lesions with both epithelial and stromal cells. The typical endometrial gland structure, including highly cylindrical epithelium, was also evident (Fig. 8F). To further explore the role of ALKBH5 in EMs progression, small interfering RNA (siRNA) targeting ALKBH5 (si-ALKBH5) or control siRNA (si-Control) was administered to model mouse by intraperitoneal injection three times a week for 4 wk. The mouse in both groups was euthanized 4 wk after the indicated treatment, and the ectopic lesion tissues were harvested. As shown in Fig. 8, C–E, different average cyst volumes and lesion weights were observed between the two treatment groups. Compared with the vehicle control, si-ALKBH5 treatment significantly inhibited the formation of abdominal wall endometriotic lesions (Fig. 8B). The immunostaining results demonstrated a substantial reduction in the expression of ALKBH5, ATG12, and LC3 signals in the si-ALKBH5-treated mouse endometriotic lesions (Fig. 8F). The Western blotting results also revealed significantly lower expression levels of ALKBH5, ATG12, and LC3 in the si-ALKBH5-treated group than in the control group (Fig. 8, G and H). In summary, these findings highlight the crucial role of ALKBH5 in the development of endometriosis, as the inhibition of ALKBH5 function prevents the formation of endometriosis and leads to the regression of endometriotic lesions.

The pathological mechanism of endometriosis remains unclear, and current therapeutic strategies are limited to surgical interventions and pharmacotherapy. Pharmacotherapeutic options include hormone therapy and nonsteroidal anti-inflammatory drugs for pain management. However, these treatments do not provide a definitive cure for the disorder. Therefore, revealing the precise mechanisms underlying EMs and developing novel potential therapeutic targets are urgently needed for the treatment of EMs. Increasing evidence has indicated that lncRNAs are closely associated with the pathogenesis of ovarian endometriosis (13, 33). For example, lncRNA C8orf49 promotes endometrial stromal cell growth and metastasis by sponging miR-1323 in endometriosis (34). LINC01116 can promote the proliferation and migration of endometrial stromal cells by targeting FOXP1 by sponging miR-9-5p (35). These reports demonstrate the important role of lncRNAs in endometriosis progression. Notably, lncRNA UBOX5-AS1 promotes epithelial-mesenchymal transition and invasion of endometrial epithelial cells under hypoxic conditions in endometriosis (16). In the present study, we found that lncRNA UBOX5-AS1 is highly expressed in both ectopic endometrial stromal cells and ovarian endometriosis tissue samples. Loss-of-function experiments revealed that lncRNA UBOX5-AS1 overexpression enhances the proliferation, migration, and invasion of endometrial stromal cells. In addition, lncRNA UBOX5-AS1 overexpression increased the protein expression levels of LC3 and ATG12, suggesting that lncRNA UBOX5-AS1 is a positive regulator of autophagy. Autophagy is the process by which cells self-consume and degrade cellular components under unfavorable conditions, ultimately allowing them to continue to survive; however excessive autophagy can lead to cell death. We previously reported that hypoxia-inducible factor (HIF)-1α promotes endometrial stromal cells invasion and survival by increasing autophagy (11, 13). In addition, autophagy contributes to hypoxia-induced Epithelial-mesenchymal transition (EMT) and the invasion of endometrial epithelial cell in endometriosis (12). These findings indicate that the abnormal activation of autophagy is involved in the pathogenesis of ovarian endometriosis. However, the mechanism of autophagy activation in patients with ovarian endometriosis remains unclear. The results of this study revealed that lncRNA UBOXA-AS1 stimulated HESCs with increased levels of autophagy and that lncRNA UBOXA-AS1 positively regulated cell autophagy. Overall, we speculate that lncRNA UBOX5-AS1 participates in the progression of endometriosis at multiple levels, especially in terms of autophagy. With the development of epigenetics and molecular biology, novel m6A methylation modifications have attracted increasing attention. As an indispensable form of epigenetic modification, m6A is suggested to be an essential regulator of female reproductive diseases (36–38). However, the functions and molecular mechanisms of m6A in the modification of lncRNAs in ovarian endometriosis remain largely unknown. Using a bioinformatic analysis tool based on published m6A or MeRIP-Seq data, we detected a remarkable m6A peak and sites in the sequence of lncRNA UBOXA-AS1. Our previous study demonstrated that m6A enrichment is decreased in ovarian endometriosis tissues (19). Epigenetic m6A modification affects disease progression through various mechanisms, and methyltransferases, demethylases, and reading enzymes play vital roles in this process (39). Here, we observed that ALKBH5 was upregulated in both the ectopic endometrial stromal cells and ovarian endometriosis tissue samples. We also explored the functional roles of ALKBH5 in EMs and reported that the upregulation of ALKBH5 could promote autophagy, proliferation, and invasion in endometrial stromal cells both in vitro and in vivo, which is consistent with the results of previous studies (26, 27, 40, 41), indicating that ALKBH5 promotes several human diseases. Furthermore, ALKBH5 promoted lncRNA UBOX5-AS1 expression. Our study also revealed the clinical significance of ALKBH5, lncRNA UBOX5-AS1, and autophagy in EMs tissues. ALKBH5 functions as a well-known demethylase to reverse m6A methylation and plays a crucial role in several pathophysiological processes. For instance, ALKBH5 has been shown to drive immune suppression and tumorigenesis in colorectal cancer via the m6A-AXIN2-Wnt-DKK1 axis (42). ALKBH5 protects against cardiac rupture after myocardial infarction by promoting the stability of ErbB4 mRNA and the degradation of ST14 mRNA via m6A demethylation (43). Moreover, knockdown of lncRNA UBOX5-AS1 partially impeded the effects caused by ALKBH5 overexpression, suggesting that ALKBH5 promoted the autophagy and progression of EMs by stabilizing lncRNA UBOX5-AS1 expression. However, the precise mechanism underlying the ability of ALKBH5 to regulate lncRNA UBOX5-AS1 remains unclear. Recently, studies have shown that m6A modification can regulate the metabolism of lncRNAs. For example, in esophageal squamous cell carcinoma (ESCC), m6A demethylation of lncRNA LINC00022 by FTO contributes to the growth of ESCC tumors in vivo (44). In glioblastoma, METTL3-mediated m6A modification of LINC00839 enhances its expression through stabilizing its RNA transcript in a YTHDF2-dependent manner (45), and our findings also clarify the potential m6A-lncRNA interaction in ovarian endometriosis. Here, we demonstrated that lncRNA UBOX5-AS1 was notably enriched in the ALKBH5-bound fraction. Our results revealed that lncRNA UBOX5-AS1 can be modified by ALKBH5 and that ALKBH5 upregulation promotes the stability and expression of lncRNA UBOX5-AS1. These results were supported by the MeRIP-qPCR results, which revealed that the m6A methylation levels of lncRNA UBOX5-AS1 were increased when ALKBH5 was ectopically overexpressed. Overall, we observed that ALKBH5 blocked m6A modification of lncRNA UBOX5-AS1 to increase its expression and stability in endometrial stromal cells. In conclusion, our work demonstrated that the m6A demethylase ALKBH5-induced lncRNA UBOX5-AS1 promotes autophagy and progression in ovarian endometriosis (Fig. 9). Our research is the first investigation of the ALKBH5/lncRNA UBOX5-AS1 axis in ovarian endometriosis. More importantly, our findings provide a better understanding of the functional roles of ALKBH5 and m6A methylation in ovarian endometriosis, and may reveal a range of new therapeutic targets involving ALKBH5 and lncRNA UBOX5-AS1, especially for the treatment of patients with ovarian endometriosis. ETHICAL APPROVALS Approval by Ethics Committee: The Research Ethics Committee of Tongji Medical College, Huazhong University of Science (IORG 140 No: IORG0003571). Human rights statements and informed consent: All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and its later amendments. Informed consent was obtained from all patients for inclusion in the study. DATA AVAILABILITY All data are available in the article or its supporting information, further inquiries can be directed to the corresponding authors. SUPPLEMENTAL MATERIAL Supplemental Figs. S1–S3 and Supplemental Tables S1–S3: https://doi.org/10.6084/m9.figshare.27998042.v1. GRANTS This work was supported by grants from the National Natural Science Foundation of China under Grant Nos. 82001524 (to Hengwei Liu) and 81974242, U20A20349, and 82371681 (to Yi Liu), the Natural Science Foundation of Hubei Province under Grant Nos. 2020CFB310 (to Hengwei Liu) and 2022CFB497 (to Xiaoli Wang). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS H.L. and Y.L. conceived and designed research; J.L. and H.L. performed experiments; J.L., X.W., and Y.X. analyzed data; J.L. and H.L. drafted manuscript; H.L., J.L., L.Z., X.D., Xiuping Wang, Xiwen Wang, and Y.L. edited and revised manuscript; J.L., H.L., Y.X., and Y.L. approved final version of manuscript. SUPPLEMENTAL MATERIAL Supplemental Figs. S1–S3 and Supplemental Tables S1–S3: https://doi.org/10.6084/m9.figshare.27998042.v1. ACKNOWLEDGMENTS We thank all patients who agreed to participate in this study and all colleagues who helped with this research.

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