{"paper_id":"b380b866-cc82-4bed-b540-ad675ebcea5e","body_text":"Front. Biosci. (Landmark Ed) 2024; 29(12): 421\nhttps://doi.org/10.31083/j.fbl2912421\nCopyright: © 2024 The Author(s). Published by IMR Press.\nThis is an open access article under the CC BY 4.0 license .\nPublisher’s Note: IMR Press stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.\nOriginal Research\nDownregulated METTL3 Accumulates TERT Expression that Promote\nthe Progression of Ovarian Endometriosis\nFang Li1,†, Hua Tao2,†, Yini Wei1,3,†, Ru Meng 1, Y ushan Li1, Lifang Nie 1, Y u Zhang2,3,*,\nJinjun Chang1,*\n1Department of Gynecology, Jincheng Hospital Affiliated to Changzhi Medical College, Jincheng People’s Hospital, 048026 Jincheng, Shanxi, China\n2Department of Gynecology, Liuzhou Maternity and Child Healthcare Hospital, 545001 Liuzhou, Guangxi, China\n3State Key Laboratory of Ultrasound in Medicine and Engineering, College of Biomedical Engineering, Chongqing Medical University, 400016\nChongqing, China\n*Correspondence: glzhangyu@126.com (Y u Zhang);changjinjun2403@126.com (Jinjun Chang)\n†These authors contributed equally.\nAcademic Editor: Marina Ivanišević\nSubmitted: 2 July 2024 Revised: 12 September 2024 Accepted: 23 September 2024 Published: 18 December 2024\nAbstract\nBackground: Endometriosis is a complicated and enigmatic disease that significantly diminishes the quality of life for women affected\nby this condition. Increased levels of human telomerase reverse transcriptase ( TERT) mRNA and telomerase activity have been found in\nthe endometrium of these patients. However, the precise function of TERT in endometriosis and the associated biological mechanisms\nremain poorly understood. Methods: We analyzed TERT expression in ectopic endometrial (EC), eutopic endometrial (EU), and normal\nendometrial (NC) tissues. Human endometrial stromal cells (HESCs) were used to study the effects of TERT depletion and knockdown\non cell behavior. We also assessed methyltransferase-like 3 (METTL3)-mediated N6-methyladenosine (m6A) modification in TERT\ntranscripts and its impact on mRNA stability and cell functions. Results: The current results indicate that TERT expression is elevated\nin EC tissue compared to both EU and NC. Depletion of TERT suppressed the proliferation and migration of HESCs, while TERT\noverexpression had the opposite effect. We found high levels of METTL3-mediated m6A modification in TERT transcripts, particularly\nin the coding sequence region, resulting in increased translation. However, EC tissues had lower m6A levels due to the downregulation\nof METTL3. Mechanistically, m6A modification mediated by METTL3 negatively regulates the stability of TERT mRNA in a YTH N6-\nmethyladenosine RNA binding protein 2 (YTHDF2)-dependent manner. Furthermore, METTL3 negatively regulated the proliferation\nand migration of HESCs. Conclusions: Together, our study identified a new molecular mechanism that underlies the pathogenesis of\nendometriosis. Inhibition of m6A modification and of the METTL3/TERT axis may enhance cellular proliferation and migration, thereby\ncontributing to the progression of endometriosis.\nKeywords: endometriosis; TERT; METTL3; YTHDF2\n1. Introduction\nEndometriosis is defined by the occurrence of tis-\nsue similar to the endometrium located outside the uterus\n[1], and it is linked to persistent pelvic discomfort and fe-\nmale infertility. This condition adversely impacts the pa-\ntients’ quality of life, sexual function, and personal rela-\ntionships [2–5]. More than 200 million women worldwide\nare affected by this condition, representing over 10% of fe-\nmales of reproductive age [6]. Ovarian endometriosis is the\nmost prevalent form of endometriosis [ 7]. Monthly, the ec-\ntopic endometrial tissue on the ovary undergoes continu-\nous proliferation, shedding, and bleeding, which progres-\nsively accumulates and leads to the formation of an ovar-\nian endometriotic cyst, commonly referred to as a choco-\nlate cyst. Ovarian endometriosis is particularly challeng-\ning to manage due to its unique pathophysiological char-\nacteristics. The formation of endometriomas often leads to\novarian damage, including inflammation, fibrosis, and scar-\nring, which can severely impact ovarian reserve and func-\ntion [8]. Moreover, ovarian endometriosis is frequently as-\nsociated with more severe disease presentations, including\ndeep infiltrating endometriosis and extensive pelvic adhe-\nsions, which complicate both clinical management and sur-\ngical intervention [9]. Despite the high prevalence and sig-\nnificant impact on quality of life, the underlying molecular\nmechanisms driving ovarian endometriosis remain poorly\nunderstood. Despite being a non-cancerous condition, en-\ndometriosis exhibits features reminiscent of malignancy,\nincluding abnormal cell migration and invasion. These fac-\ntors contribute to endometriosis and to its recurrence af-\nter surgery. Despite extensive research over the past few\ndecades into the multifactorial causes of endometriosis [10–\n13], the underlying mechanism remains unclear.\nTelomerase plays a vital role in preserving telomere\nlength and ensuring cellular immortality [ 14–16]. With\nthe exception of hematopoietic stem cells and endometrial\ncells, telomerase is generally not expressed in normal so-\nmatic cells. Nonetheless, telomerase activity is present in\n\nvarious cancers and is linked to their capacity for limit-\nless replication. Human telomerase reverse transcriptase\n(hTERT) is a catalytic protein subunit essential for telom-\nerase function. A significant relationship has been noted be-\ntween hTERT mRNA levels and telomerase activity across\ndifferent tissues [ 17,18], with hTERT mRNA serving as a\nkey factor in regulating telomerase activity [ 19]. Telom-\nerase activity and hTERT mRNA levels in endometrial\ncancer are significantly higher than in the normal cycling\nendometrium [ 20]. Interestingly, telomerase activity and\nhTERT mRNA expression in patients with endometriosis\ncorrelate with the proliferative potential of the endometrium\n[21]. These findings indicate that TERT plays an essential\nrole in the adhesion of endometrial cells, and that abnormal\nTERT expression may contribute to the pathological pro-\ncesses involved in endometriosis. However, this connec-\ntion has yet to be investigated, and the upstream regulatory\nmechanisms of TERT remain unknown.\nN6-methyladenosine (m6A) is among the most com-\nmon modifications found in RNA [ 22,23]. Like DNA\nmethylation, m6A exhibits dynamic and reversible regula-\ntory properties. This modification is added by a methyl-\ntransferase complex comprising two primary subunits [ 24].\nDemethylation of m6A requires the action of fat mass\nand obesity-associated protein (FTO) [ 25] and alkB ho-\nmolog 5 (ALKBH5) [ 26]. The equilibrium between m6A\nmethyltransferases and demethylases is essential for the dy-\nnamic regulation of m6A. Increasing evidence indicates\nthat m6A plays a significant role in various disease pro-\ncesses, particularly in cancer metastasis [ 27]. Conversely,\nreduced methyltransferase-like 14 (METTL14) expression\npromotes hepatocellular carcinoma cell metastasis by in-\nfluencing the processing of m6A-dependent primary mi-\ncroRNA (pri-miRNA) [ 28]. However, the precise role of\nm6A and its associated mechanisms in the development of\nendometriosis remains elusive and requires further inves-\ntigation. This study was to explore the influence of m6A\nmodification on the upregulation of TERT expression ob-\nserved in ectopic endometrial tissue. A better understand-\ning of the epigenetic regulation of TERT via m6A modifi-\ncation may shed light on the pathogenesis of endometriosis\nand identify potential therapeutic targets for this condition.\nThis study provides evidence that reduced m6A mod-\nification is a key driving factor for the aberrant expression\nof TERT in endometriosis. Our findings highlight the sig-\nnificance of methyltransferase-like 3 (METTL3)/TERT in\nthe development of endometriosis and suggest that TERT\ncould serve as a novel target in the diagnosis and therapy of\nthis condition.\n2. Methods\n2.1 Patient Enrollment and Tissue Collection\nIndividuals with ovarian endometriosis were chosen\nfrom the Obstetrics and Gynecology Department at Li-\nuzhou Maternal and Child Health Hospital. Post-surgery,\nthe endometrial tissues were categorized into eutopic en-\ndometrial (EU) and ectopic endometrial (EC). EU samples\nwere sourced from the endometrial lining within the uterus,\nwhile EC samples were taken from the ovarian tissue. We\nincluded women aged 18–45 with confirmed ovarian en-\ndometriosis and excluded those with malignancies, autoim-\nmune diseases, other gynecological conditions, recent hor-\nmone therapy (within six months), or systemic diseases.\nTo control for age, we focused on women aged 18–45 and\nensured all samples were collected during the proliferative\nphase of the menstrual cycle without recent hormone treat-\nment to minimize hormonal and systemic effects. Normal\nendometrium (normal control; n = 8) was obtained dur-\ning the surgical operation. Human tissue samples were\nobtained with patients’ or their families/legal guardians’\nwritten informed consent, in accordance with the Declara-\ntion of Helsinki guidelines. The research received approval\nfrom the Ethics Committee of Liuzhou Maternal and Child\nHealth Hospital (KS-KY -2020-073).\n2.2 Cell Culture\nHuman endometrial stromal cells (HESCs) were\nsourced from Otwo Biotech (HTX2487-2, Shenzhen,\nChina) and cultured with DMEM as previously [ 29]. The\nculture medium was enriched with 1.5 g/L sodium bicar-\nbonate (144-55-8, Sigma-Aldrich, St. Louis, MO, USA),\n1% recombinant Human Insulin (ITS)+ Premix (354352,\nCorning, Corning, NY , USA), 500 ng/mL puromycin, and\n10% charcoal-stripped fetal bovine serum (F6765, Sigma-\nAldrich), and kept in a humidified incubator at 37 °C in\na 5% CO 2 environment. All cell lines were validated by\nshort tandem repeat (STR) profiling and tested negative for\nmycoplasma. 5-Azacytidine (5-aza-dC) (HY -10586) and\nSuberoylanilide Hydroxamic Acid (SAHA) (HY -10221)\nwere obtained from MCE (Middlesex County, NJ, USA),\nwhile sodium butyrate (NaB) (S1999) was acquired from\nSelleck. The expression of TERT in HESC cells was as-\nsessed by real-time quantitative PCR (qPCR) after treat-\nment for 12 hours with SAHA (5 µM) or NaB (5 mM), or\nwith 5 µM 5-Aza-dC for 48 hours.\n2.3 Lentiviral Transduction\nTo overexpress TERT and METTL3 in HESCs, hu-\nman TERT and METTL3 were synthesized and cloned\ninto the pLVX plasmid vector (General Biosystems, An-\nhui, China). For the knockdown of TERT and METTL3,\nlentiviruses containing small interfering RNAs (siRNAs)\ntargeting TERT (5′-AAAAA TGTGGGGTTCTTCCAA-\n3′), METTL3 (5′-TCTAACTCAGGA TCTGTAGCT-3′), or\na nonsense siRNA (5 ′-CAGCCA TCAACTCAGA TTGTT-\n3′) were constructed by General Biosystems (Anhui,\nChina). Recombinant lentiviral vectors or control vectors,\nalong with two auxiliary packaging plasmids (pSPAX2 and\npMD2G), were co-transfected into HEK293T cells to pro-\nduce the lentiviruses, as provided by General Biosystems\n2\n\n\n(Anhui, China). After 48 hours, the supernatant was col-\nlected and used to infect HESCs with an initial multiplicity\nof infection (MOI) of 50. Transduction efficiency was as-\nsessed 48 hours post-infection using Western blot analysis\n(Supplementary Fig. 1 ).\n2.4 Immunohistochemistry\nThe retrieved tissue specimens were promptly pre-\nserved in 4% buffered formalin and subsequently pre-\npared for immunohistochemical (IHC) analysis by Service-\nbio Biotechnology Co., Ltd. (Wuhan, China). Paraffin-\nembedded slices were subjected to staining with a primary\nantibody targeting METTL3 (1:200, Proteintech, 15073-1-\nAP , San Diego, CA, USA) according to the manufacturer’s\ninstructions.\n2.5 RNA m6A Quantitative Assays\nTo assess m6A levels in total RNA samples, an\nEpiQuik m6A RNA Methylation Quantification Kit (Epi-\ngentek, P-9005-48, Farmingdale, NY , USA) was utilized.\nInitially, the RNA was applied to wells pre-coated with\nRNA high binding solution, then capture and detection an-\ntibody solutions were introduced. The m6A levels were de-\ntermined by measuring absorbance at 450 nm and calculat-\ning relative quantification.\n2.6 Cell Proliferation Assays\nCell proliferation was evaluated through the Cell\nCounting Kit-8 (CCK-8) assay (MCE, HY -K0301). Cells\nwere seeded in a 96-well plate and treated as needed. Af-\nter treatment, 10 µL of CCK-8 solution was added to each\nwell and incubated at 37 ℃ for 1–4 hours. Absorbance at\n450 nm was then measured using a microplate reader to\nassess cell proliferation and viability. For 5-ethynyl-2’ -\ndeoxyuridine (EdU) staining, the culture medium was sup-\nplemented with 10 µM EdU reagent (Beyotime Biotechnol-\nogy, C0078S, Shanghai, China) and incubated for 3 hours\nat 37 °C. Cells were then stained with Hoechst 33342 as\nper the manufacturer’s instructions and examined under a\nfluorescence microscope.\n2.7 Transwell Assays\nCell invasion and migration were assessed using Tran-\nswell chambers (Corning, Corning, NY , USA). For inva-\nsion assays, Matrigel (Corning, 354248) was mixed with\nfetal bovine serum (FBS)-free DMEM/F12 medium at a 1:3\nratio and applied to the upper chamber. For migration as-\nsays, Matrigel was not used. Cells (8 × 104) were resus-\npended in 200 µL of serum-free DMEM/F12 and placed in\nthe upper chamber, while the lower chamber contained 600\nµL of medium with 20% FBS. After 24 hours, cells that ad-\nhered to the membrane’s underside were fixed, stained, and\nthen quantified using a microscope.\n2.8 Wound Healing Assay\nOnce cells reached 90–100% confluence, a sterilized\npipette tip (200 µL) was used to create scratches. Following\nPBS washes to remove detached cells and debris, wound\nhealing was monitored and photographed at 0 and 24 hours\npost-scratching. The wound area at each time point was\ncompared to the initial size at 0 hours, and the percentage\nof area closure was calculated.\n2.9 RIP Assay\nThe RIP assay was conducted with the Magna™ RIP\nRNA-Binding Protein Immunoprecipitation Kit (Millipore,\n17-700, Burlington, MA, USA). Cells were lysed using a\nRIP lysis buffer, and magnetic beads were conjugated with\nhuman antibodies against YTH N6-methyladenosine RNA\nbinding protein 2 (YTHDF2) (Proteintech, 24744-1-AP) or\nm6A (Synaptic Systems, 202-003, Gottingen, Germany).\nNormal mouse Immunoglobulin G (IgG) antibody (Milli-\npore, 12-370) was used as a negative control. RNA ob-\ntained with the RIP assay was assessed by RT-qPCR and\nnormalized to the input.\n2.10 Western Blotting\nTotal protein was extracted with RIPA buffer (Bey-\notime, P0013B). Following protein quantification, SDS-\nPAGE separation, and transfer onto polyvinylidene diflu-\noride (PVDF) membranes were performed. The mem-\nbranes were treated with 5% skim milk to prevent non-\nspecific binding, and then incubated with the specified an-\ntibodies: TERT (1:1000, Thermo, MA5-16034, Waltham,\nMA, USA), METTL3 (1:2000, Cell Signaling Technol-\nogy, #86132, Danvers, MA, USA.), YTHDF2 (1:2000, Ab-\ncam, ab246514, Cambridge, UK), YTHDF3 (1:2000, Ab-\ncam, ab220161), IGF2BP1 (1:2000, Abcam, ab290736),\nIGF2BP2 (1:2000, Abcam, ab124930), IGF2BP3 (1:2000,\nAbcam, ab177477), and GAPDH (1:6000, Proteintech,\n60004-1-Ig) overnight at 4 °C. Subsequent incubation with\nsecondary antibodies was followed by detection of pro-\ntein bands using ECL chemiluminescent reagent (Bey-\notime, P0018S) and densitometry analysis using ImageJ\n(version 1.41, LOCI, University of Wisconsin, Madison,\nWI, USA).\n2.11 Luciferase Reporter Assay\nThe luciferase reporter assay was carried out utilizing\nthe Dual-Luciferase Reporter Assay System. Cells were\ntransfected with a pmiGLO-based luciferase vector carry-\ning either wild-type or mutated TERT, or a control vec-\ntor, employing Lipofectamine 3000 reagent (Invitrogen,\nWaltham, MA, USA). After 24 hours, the luciferase and\nRenilla activities were measured according to the man-\nufacturer’s guidelines. For further analysis, pmirGLO-\nTERT-3′UTR-WT and pmirGLO-TERT-3 ′UTR-Mut con-\nstructs were introduced into control or shMETTL3 cells for\n24 hours, and F-luc and R-luc activities were evaluated.\n3\n\nFig. 1. TERT overexpression in endometriosis. (A,B) Protein (A) and mRNA (B) expression levels of TERT in NC, EU, and EC\ntissue. (C) Immunohistochemistry of TERT expression in the endometriosis and control groups. Scale bar: 200 µm or 80 µm. Error bars\nrepresent the mean ± SD of results from triplicate biological experiments. ** p < 0.01. TERT, telomerase reverse transcriptase; NC,\nnormal endometrial; EU, eutopic endometrial; EC, ectopic endometrial.\n2.12 mRNA Stability\nTo measure RNA stability, cells were treated with\n5 µg/mL actinomycin D (Act-D, Sigma-Aldric, #A9415).\nRNA samples were collected at various time intervals post-\ntreatment, and real-time PCR was performed to analyze\nRNA levels. The TERT mRNA half-life ( t1/2) was cal-\nculated with the formula ln2/slope, using GAPDH as the\nreference control.\n2.13 Real-Time PCR\nRNA was extracted with Trizol reagent (Invitrogen,\nA33250). For cDNA synthesis, 500 ng of mRNA was\nreverse transcribed with the PrimeScript RT Master Kit\n(Takara, Dalian, China). qPCR was performed using TB\nGreen Premix Ex Taq II (TaKaRa, RR820Q), and transcript\nlevels were determined based on the threshold cycle num-\nber. GAPDH served as the normalization control, and rel-\native expression was calculated using the 2 −∆∆Ct method.\nThe primer sequences are listed in Supplementary Table\n1.\n2.14 Statistical Analysis\nResults are expressed as mean ± SEM. The Shapiro-\nWilk test was employed to evaluate data normality. For\ncomparing two groups, an unpaired Student’s t-test was\nused for normally distributed data, while the Mann-\nWhitney test was applied for data that did not follow a nor-\nmal distribution. For multiple group comparisons, ANOV A\nwas performed with subsequent Bonferroni correction to\nadjust for multiple tests. A significance level of p < 0.05\n4\n\n\nwas adopted, and all experiments were performed indepen-\ndently. Statistical analyses were performed using Graph-\nPad Prism software (version 9.1.4, Dotmatics, Boston, MA,\nUSA).\n3. Results\n3.1 Upregulation of TERT in Ectopic Tissues\nTo explore the involvement of TERT in endometriosis,\nwe first evaluated its expression levels using Western blot-\nting and qRT-PCR across normal endometrial (NC), EU,\nand EC. Our analysis revealed a notable elevation in TERT\nexpression in EC relative to both EU and NC (Fig. 1A,B).\nNo significant variation in expression levels was detected\nbetween EU and NC. To confirm these findings, we con-\nducted immunohistochemistry (IHC) to assess TERT ex-\npression across NC, EU, and EC samples. The IHC results\nverified higher TERT expression in EC relative to NC and\nEU (Fig. 1C), supporting the results obtained from qRT-\nPCR and Western blot analyses. This indicates that in-\ncreased TERT levels may play a role in the progression of\nendometriosis.\n3.2 TERT Promotes the Proliferation and Migration of\nEndometrial Stromal Cells in Vitro\nTo explore the potential biological function of TERT\nin HESCs, lentiviruses were used to knockdown (shTERT)\nor overexpress TERT. EdU assay and CCK-8 assays were\nthen performed to evaluate cell viability following TERT\nknockdown or overexpression. TERT knockdown signifi-\ncantly reduced the proliferation of HESCs, whereas TERT\noverexpression had the opposite effect (Fig. 2A,B).\nNext, the migratory and invasive abilities of HESCs\nwith altered TERT expression were investigated using\nwound healing and transwell assays. TERT overexpression\nwas found to promote cell migration and invasion, whereas\nTERT knockdown inhibited these processes (Fig. 2C,D).\nThese findings indicate that TERT plays a crucial role\nin promoting cell proliferation and migration during en-\ndometriosis.\n3.3 Downregulation of METTL3-Mediated m6A\nModification in Endometriosis\nRecent studies demonstrated the involvement of epi-\ngenetic mechanisms in gene dysregulation. We therefore\ninvestigated whether epigenetic factors contribute to the up-\nregulation of TERT in endometriosis. TERT expression re-\nmained unchanged following the treatment of HESCs with\na DNA methyltransferase inhibitor (5-aza-dC), suggesting\nthat DNA methylation does not play a role in regulat-\ning TERT. Furthermore, the application of broad-spectrum\nhistone deacetylase (HDAC) inhibitors SAHA and NaB\ndid not affect TERT expression, indicating that histone\nacetylation is not involved in TERT regulation in HESCs\n(Supplementary Fig. 2 ).\nm6A modification in endometriosis was investigated\nby measuring m6A levels in NC, EU, and EC using a col-\norimetric method. The results showed that m6A levels\nwere lower in EC compared to NC and EU. No signifi-\ncant difference in the m6A level was observed between\nEU and NC (Fig. 3A). The expression of m6A-associated\ngenes in NC, EU, and EC was subsequently evaluated in\norder to identify the key molecules responsible for reduced\nm6A in endometriosis. Both the mRNA and protein levels\nof METTL3 were found to be downregulated in EC rela-\ntive to NC and EU (Fig. 3B–D). These findings were con-\nsistent with the lower m6A levels, suggesting a potential\nrole for METTL3 in m6A modification in endometriosis.\nMoreover, overexpression of METTL3 was found to signif-\nicantly reduce TERT mRNA and protein expression, while\nreducing METTL3 levels led to an increase in TERT ex-\npression relative to normal controls (Fig. 3E,F).\n3.4 Involvement of TERT in METTL3-Induced Cell\nProliferation, Migration and Invasion\nWe investigated how TERT influences METTL3-\ndriven cell proliferation, migration, and invasion in HESCs\nby introducing a METTL3-overexpressing lentivirus into\ncells with TERT overexpression. It was observed that ele-\nvated METTL3 levels markedly reduced cell proliferation,\nmigration, and invasion. In contrast, TERT overexpression\ncounteracted these suppressive effects (Fig. 4A–D). These\nfindings indicate that TERT plays a role in modulating the\nMETTL3-mediated inhibition of HESC proliferation, mi-\ngration, and invasion.\n3.5 TERT is Regulated by METTL3-Mediated m6A\nModification\nEmerging evidence suggests that m6A influences var-\nious aspects of RNA fate [ 30–32]. We next investigated\nthe role of m6A modification in TERT upregulation. Data\nfrom the online RNA modification resource (RMBase, http:\n//rna.sysu.edu.cn/rmbase/) indicate that multiple m6A sites\nare present within the TERT transcript ( Supplementary\nFig. 3 ). In order to validate whether TERT is a target\nof METTL3-mediated m6A modification, we performed\nMeRIP-qPCR in HESCs. A remarkable decrease in m6A\nmodification of TERT was observed when METTL3 was\nknocked down (Fig. 5A). Furthermore, the distribution of\nm6A methylation in TERT mRNA was investigated using\nfragmented RNA isolated from HESCs. The highest level\nof m6A methylation was observed in the 3 ′ untranslated\nregion (UTR) of TERT (Fig. 5B). Two m6A modification\nsites were identified within the 3 ′UTR of TERT (Fig. 5C).\nTo investigate the potential role of m6A in the 3 ′UTR, lu-\nciferase reporters containing the wild-type or mutant TERT\nm6A methylation sites were generated (Fig. 5D). A fire-\nfly luciferase reporter assay demonstrated that, in HESCs\nwith METTL3 knockdown, the luciferase activity of the re-\nporter with the wild-type site was markedly elevated com-\n5\n\nFig. 2. The impact of TERT on HESC proliferation, migration, and invasion. (A,B) The influence of TERT silencing or overex-\npression on HESC proliferation was determined by CCK-8 assay (A) and 5-ethynyl-2’ -deoxyuridine (EdU) incorporation (B) Scale bar:\n100 µm. (C) Transwell assays were conducted to evaluate migration (without matrigel) and invasion (with matrigel) 24 hours post-TERT\nmanipulation. Representative images and quantification are displayed. Scale bar: 100 µm. (D) The wound healing assay was employed\nto measure cell motility at 0 and 24 hours, with representative images and quantitative data provided. Scale bar: 100 µm. Error bars\ndenote the mean ± SD from 3 independent experiments. * p < 0.05, **p < 0.01. HESC, Human endometrial stromal cell; CCK-8, Cell\nCounting Kit-8; shNC, short hairpin negative control.\n6\n\n\nFig. 3. METTL3 decreases m6A levels in endometriosis. (A) m6A content in NC, EU, and EC tissue. (B) mRNA expression of m6A-\nrelated genes measured by quantitative Reverse Transcription PCR (qRT-PCR). (C) METTL3 protein levels assessed through Western\nblotting. (D) METTL3 localization in tissues identified via Immunohistochemistry (IHC). Scale bar: 200 µm or 80 µm. (E,F) TERT\nmRNA levels evaluated by qRT-PCR (E) and Western blotting (F). Error bars indicate the mean± SD from three independent experiments.\n*p < 0.05, **p < 0.01. METTL3, methyltransferase-like 3; m6A, N6-methyladenosine.\n7\n\nFig. 4. METTL3 regulates HESC proliferation, migration, and invasion via TERT. (A,B) The EdU (A) and CCK-8 assay (B)\nwere used to evaluate the effect of negative control and METTL3-overexpressing vector on HESC proliferation, both in the absence or\npresence of TERT overexpression. Scale bar: 100 µm. (C) Transwell assays were performed 24 h after transfection. Scale bar: 100 µm.\n(D) Changes in cell motility were assessed through wound healing assays at 0 and 24 h. Representative images and quantification are\npresented. Scale bar: 100 µm. Error bars represent the mean ± SD of triplicate biological experiments. ** p < 0.01.\n8\n\n\npared to that observed in control cells. However, muta-\ntions in either one or both of the TERT m6A motifs abol-\nished the increased luciferase activity observed in cells with\nMETTL3 knockdown (Fig. 5E). These findings suggest that\nm6A methylation in the TERT 3 ′UTR is responsible for\nmRNA stability. Additionally, TERT mRNA expression\nwas higher in cells co-transfected with METTL3 and mu-\ntant TERT-3′UTR sites (TERT-3′UTR-MUTs) compared to\ncells with the wild-type site (TERT-3 ′UTR-WT) (Fig. 5F).\nFurthermore, the mRNA stability of TERT-3′UTR-WT was\ngreater in control cells than in cells with METTL3 overex-\npression, and the mutation in TERT-3 ′UTR abolished this\ndifference (Fig. 5G,H). These results indicate that m6A\nin the TERT 3 ′UTR is crucial for mRNA stability, possi-\nbly due to its impact on the secondary structure of TERT\nmRNA.\n3.6 YTHDF2 Promotes the Degradation of TERT mRNA\nthrough an m6A-Dependent Mechanism\nTo regulate gene expression, m6A modification is\nperformed by m6A writers and requires recognition by\nm6A readers [ 33,34]. m6A readers, such as YTHDF2,\nYTHDF3, and IGF2BP1~3, can modulate mRNA stabil-\nity [ 35,36]. The present study found that YTHDF2, but\nnot YTHDF1, YTHDF3, or IGF2BP1/2/3, showed signif-\nicant binding to TERT mRNA in HESCs (Fig. 6A). Fur-\nthermore, the interaction between YTHDF2 and TERT was\nreduced in HESCs with METTL3 knockdown relative to\ncontrol cells (Fig. 6B). Stability assays of mRNA indicated\nthat TERT mRNA had an extended half-life in cells with\nYTHDF2 knockdown, while it was notably shorter in cells\nwith YTHDF2 overexpression (Fig. 6C,D). Consistent with\nthese findings, YTHDF2 negatively regulated TERT ex-\npression in HESCs (Fig. 6E,F). Additionally, the expression\nlevel of TERT mRNA in cells co-transfected with YTHDF2\nand mutant TERT-3′UTR sites (TERT-3′UTR-MUTs) was\nhigher than in cells co-transfected with YTHDF2 and\nthe wild-type site (TERT-3 ′UTR-WT) (Fig. 6G). These\nfindings demonstrate that YTHDF2 recognizes METTL3-\nmethylated TERT mRNA and facilitates its decay (Fig. 7).\n4. Discussion\nEndometriosis is a multifaceted and poorly understood\ncondition that profoundly affects those who suffer from\nit. Although its origins are thought to be multifactorial,\nthe precise functional and biological processes driving its\nonset remain largely elusive. V arious studies have exam-\nined genes with increased expression in the endometrium\nof affected individuals. In this study, we explored the in-\nvolvement of human TERT and m6A modification in en-\ndometriosis to uncover a potential new molecular mecha-\nnism underlying its development.\nHuman TERT is the catalytic subunit of telomerase\nand plays a crucial role in its activity [ 37]. The classi-\ncal functions of TERT are to activate telomerase activity,\nsynthesize telomere DNA, maintain telomere stability, and\nconfer cells with the potential for unlimited proliferation.\nThese functions are closely related to tumorigenesis and\ndevelopment, with TERT being highly expressed in over\n90% of human malignancies [ 38]. Besides the telomerase\npathway, recent studies have also shown that TERT partic-\nipates in the regulation of gene expression and affects tu-\nmor cell invasion and gene regulation through telomerase-\nindependent mechanisms. Overexpression of TERT re-\nmarkably increased the in vitro invasive ability of cells, and\nthat hTERT expression in gastric cancer tissue was closely\nassociated with tumor progression. High expression of\nTERT was also shown to enhance the invasive ability of tu-\nmor cells in cervical cancer, osteosarcoma and breast cancer\n[39]. This was accompanied by upregulation of metallopro-\nteinase. Although endometriosis is not a malignant disease,\nmany of its clinical and biological manifestations resemble\nthe characteristics of malignant tumors, including abnormal\ncell proliferation, invasion, diffusion, and even metastasis.\nSimilar to its role in the invasion and metastasis of tumors,\nhTERT may therefore also have an important role in the in-\nvasion of ectopic endometrial cells. Our findings revealed\nthat TERT expression was upregulated in ectopic endome-\ntrial (EC) tissue compared to eutopic endometrial (EU) and\nnormal endometrial (NC) tissue. This observation suggests\nthe potential involvement of TERT in the aberrant cellular\nbehaviors associated with endometriosis. To further elu-\ncidate the function of TERT, we conducted experiments\nto investigate the impact of its depletion in HESCs. We\nfound that suppression of TERT expression led to a signif-\nicant decrease in the proliferation, migration, and invasion\nof HESCs, whereas TERT overexpression had the opposite\neffect. This result suggests that TERT plays a crucial role\nin promoting the aggressive behavior of endometrial cells\nin endometriosis.\nEpigenetic processes have been linked to gene expres-\nsion dysregulation across various diseases, including can-\ncers and developmental disorders [ 40,41]. This study ex-\nplored whether epigenetic mechanisms contribute to the el-\nevated expression of TERT seen in endometriosis. We first\nexamined whether DNA methylation and histone acety-\nlation were involved in TERT expression, but found no\nsignificant changes in expression after treatment with in-\nhibitors targeting these epigenetic modifications. This led\nus to investigate the potential role of m6A modification,\nthe most frequent RNA modification, in the regulation of\nTERT expression. Our results showed lower m6A levels\nin endometriotic tissue compared to NC and EU. Previ-\nous research has indicated that decreased expression of the\nm6A methyltransferase METTL3 facilitates endometriosis\ndevelopment. METTL3 was found to be downregulated in\nendometriotic stromal cells, which enhances their migration\nand invasion through the METTL3/m6A/miR126 pathway\n[42]. Our study further confirmed the reduced expression of\nMETTL3 in endometriotic cells. Additionally, we observed\n9\n\nFig. 5. METTL3-driven m6A modification regulates TERT expression. (A,B) Methylated RNA immunoprecipitation (MeRIP)-\nqPCR analysis of relative enrichment of m6A on TERT. (C) A diagram illustrates the locations and mutations of m6A sites within the\nTERT 3 ′UTR, with “mut” denoting mutant and “chr” referring to chromosome. (D) A schematic shows the mutations introduced in\nthe 3 ′UTR to examine the impact of m6A on TERT expression. (E) The relative luciferase activity was measured for in control or\nshMETTL3 HESCs. (F) TERT-3 ′UTR-WT or TERT-3 ′UTR-DMut was transfected into control or shMETTL3 HESCs for 24 hours,\nfollowed by qPCR analysis to determine TERT mRNA expression levels. (G,H) Following 24-hour transfections of TERT-3 ′UTR-\nWT (G) or TERT-3 ′UTR-DMut (H) into control or shMETTL3 cells, the cells were treated with actinomycin D (5 µg/mL) for various\ndurations. TERT mRNA levels were subsequently measured by qPCR. Error bars indicate the mean ± SD from triplicate experiments.\n**p < 0.01; ns. indicates not significant. IgG, Immunoglobulin G; WT, wild type.\n10\n\n\nFig. 6. YTHDF2 regulates the decay of TERT mRNA in an m6A-dependent fashion. (A) TERT mRNA was examined using RIP-\nqPCR with antibodies specific to YTHDF2, YTHDF3, and IGF2BP1~3. (B) The binding of YTHDF2 to TERT mRNA was assessed\nin control and shMETTL3 cells via RIP-qPCR. (C,D) The degradation rate of TERT mRNA was analyzed at various time points after\ntreating with actinomycin D (5 µg/mL). (E,F) The levels of TERT protein (E) and TERT mRNA (F) were measured following the\nsuppression or overexpression of YTHDF2 in HESCs. (G) TERT-3 ′UTR-WT or TERT-3 ′UTR-DMut was transfected into control or\nYTHDF2-overexpressing HESCs for 24 hours, after which TERT mRNA expression was quantified using qPCR. Error bars denote the\nmean ± SD from triplicate biological experiments. ** p < 0.01. YTHDF2, YTH N6-methyladenosine RNA binding protein 2.\n11\n\nFig. 7. Graphical representation of the roles of METTL3 and TERT in endometriosis. METTL3 modifies TERT mRNA through\nm6A, while YTHDF2 mediates the decay of TERT mRNA in an m6A-dependent manner.\nthat overexpression of METTL3 led to decreased TERT ex-\npression, while METTL3 knockdown resulted in increased\nTERT levels. These results imply that METTL3-mediated\nm6A modification plays a role in regulating TERT expres-\nsion in endometriosis. Moreover, the impact of METTL3-\nmediated m6A modification on TERT mRNA stability ap-\npears to be YTHDF2-dependent, suggesting this regulatory\naxis is involved in the pathogenesis of endometriosis.\nOur study highlights a novel molecular mechanism in-\nvolving METTL3-mediated m6A modification of TERT,\nwhich could have significant clinical implications. By elu-\ncidating the role of TERT and METTL3 in endometriosis,\nour findings provide a foundation for developing targeted\ntherapies aimed at modulating this pathway. Inhibiting\nMETTL3 or altering m6A modification could potentially\noffer new treatment options for managing endometriosis,\nimproving patient outcomes, and enhancing quality of life.\nHowever, translating these insights into clinical practice\nwill require additional research and validation in clinical\nsettings. Despite the valuable insights provided by our\nstudy, several limitations must be acknowledged. Our\nfindings are primarily based on in vitro experiments using\nHESCs and tissue samples. The absence of in vivo ani-\nmal model studies limits our ability to fully understand how\nTERT and METTL3-mediated m6A modification impact\nendometriosis in a living organism. Further research us-\ning animal models is needed to validate these findings and\nassess their relevance to human disease. While our study\nidentifies a potential role of METTL3-mediated m6A mod-\nification in regulating TERT mRNA stability and HESC cell\nbehavior, the precise mechanisms underlying these interac-\ntions remain to be fully elucidated. Additional research is\nneeded to explore how METTL3 influences TERT function\nand to identify other potential regulatory factors involved\nin this pathway.\n12\n\n\n5. Conclusions\nOur study reveals a new molecular mechanism in en-\ndometriosis, suggesting that dysregulation of m6A modifi-\ncation through the METTL3/TERT axis enhances the mi-\ngration and invasion of endometriotic cells. This pathway\nmay offer potential therapeutic targets for novel treatments.\nHowever, the study’s limitations include its reliance on in\nvitro cell lines, necessitating further validation in animal\nmodels. Additionally, the interaction between TERT and\nm6A modification in endometriosis needs further investi-\ngation. In summary, our findings highlight the role of the\nMETTL3/TERT axis in endometriosis and pave the way for\nfuture research and targeted therapies.\nAvailability of Data and Materials\nThe datasets used and/or analyzed during the current\nstudy are available from the corresponding author on rea-\nsonable request.\nAuthor Contributions\nConceptualization: JJC, YZ and FL; data acquisition:\nHT, YNW, RM, YSL, LFN and FL; data analysis: JJC, YZ,\nHT, YNW and FL; original draft preparation: JJC, YZ, HT,\nYNW and FL; review and editing: JJC, YZ, HT, YNW,\nRM, YSL, LFN and FL; all authors revised and agreed the\nfinal version of the manuscript. All authors have partici-\npated sufficiently in the work and agreed to be accountable\nfor all aspects of the work.\nEthics Approval and Consent to Participate\nHuman tissue samples were obtained with patients’\nor their families/legal guardians’ written informed consent,\nin accordance with the Declaration of Helsinki guidelines.\nThe research received approval from the Ethics Committee\nof Liuzhou Maternal and Child Health Hospital (KS-KY -\n2020-073).\nAcknowledgment\nWe would like to express our sincere gratitude to all\nindividuals and organizations who have contributed to this\nresearch. Special thanks to Y anjun Zheng for their invalu-\nable guidance and support throughout this study.\nFunding\nThis work was funded by the Guangxi Science\nand Technology Plan Project (Guangxi Clinical Research\nCenter for Obstetrics and Gynecology), grant #GuiKe\nAD22035223, and Liuzhou Science and Technology Plan\nProject, grant #2020NBAB0825. Also was supported\nby Foundation of State Key Laboratory of Ultrasound in\nMedicine and Engineering [Grant No.2021KFKT020].\nConflict of Interest\nThe authors declare no conflict of interest.\nSupplementary Material\nSupplementary material associated with this article\ncan be found, in the online version, at https://doi.org/10.\n31083/j.fbl2912421.\nReferences\n[1] Ochoa Bernal MA, Fazleabas A T. The Known, the Unknown\nand the Future of the Pathophysiology of Endometriosis. Inter-\nnational Journal of Molecular Sciences. 2024; 25: 5815.\n[2] Allaire C, Bedaiwy MA, Y ong PJ. Diagnosis and management\nof endometriosis. Canadian Medical Association Journal. 2023;\n195: E363–E371.\n[3] Li Y , Liu H, Y e S, Zhang B, Li X, Y uan J, et al. The effects of\ncoagulation factors on the risk of endometriosis: a Mendelian\nrandomization study. 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