Downregulated METTL3 Accumulates TERT Expression that Promote the Progression of Ovarian Endometriosis

Background: Endometriosis is a complicated and enigmatic disease that significantly diminishes the quality of life for women affected by this condition. Increased levels of human telomerase reverse transcriptase ( TERT) mRNA and telomerase activity have been found in the endometrium of these patients. However, the precise function of TERT in endometriosis and the associated biological mechanisms remain poorly understood. Methods: We analyzed TERT expression in ectopic endometrial (EC), eutopic endometrial (EU), and normal endometrial (NC) tissues. Human endometrial stromal cells (HESCs) were used to study the effects of TERT depletion and knockdown on cell behavior. We also assessed methyltransferase-like 3 (METTL3)-mediated N6-methyladenosine (m6A) modification in TERT transcripts and its impact on mRNA stability and cell functions. Results: The current results indicate that TERT expression is elevated in EC tissue compared to both EU and NC. Depletion of TERT suppressed the proliferation and migration of HESCs, while TERT overexpression had the opposite effect. We found high levels of METTL3-mediated m6A modification in TERT transcripts, particularly in the coding sequence region, resulting in increased translation. However, EC tissues had lower m6A levels due to the downregulation of METTL3. Mechanistically, m6A modification mediated by METTL3 negatively regulates the stability of TERT mRNA in a YTH N6- methyladenosine RNA binding protein 2 (YTHDF2)-dependent manner. Furthermore, METTL3 negatively regulated the proliferation and migration of HESCs. Conclusions: Together, our study identified a new molecular mechanism that underlies the pathogenesis of endometriosis. Inhibition of m6A modification and of the METTL3/TERT axis may enhance cellular proliferation and migration, thereby contributing to the progression of endometriosis.

endometriosis; TERT; METTL3; YTHDF2 1. Introduction Endometriosis is defined by the occurrence of tis- sue similar to the endometrium located outside the uterus [1], and it is linked to persistent pelvic discomfort and fe- male infertility. This condition adversely impacts the pa- tients’ quality of life, sexual function, and personal rela- tionships [2–5]. More than 200 million women worldwide are affected by this condition, representing over 10% of fe- males of reproductive age [6]. Ovarian endometriosis is the most prevalent form of endometriosis [ 7]. Monthly, the ec- topic endometrial tissue on the ovary undergoes continu- ous proliferation, shedding, and bleeding, which progres- sively accumulates and leads to the formation of an ovar- ian endometriotic cyst, commonly referred to as a choco- late cyst. Ovarian endometriosis is particularly challeng- ing to manage due to its unique pathophysiological char- acteristics. The formation of endometriomas often leads to ovarian damage, including inflammation, fibrosis, and scar- ring, which can severely impact ovarian reserve and func- tion [8]. Moreover, ovarian endometriosis is frequently as- sociated with more severe disease presentations, including deep infiltrating endometriosis and extensive pelvic adhe- sions, which complicate both clinical management and sur- gical intervention [9]. Despite the high prevalence and sig- nificant impact on quality of life, the underlying molecular mechanisms driving ovarian endometriosis remain poorly understood. Despite being a non-cancerous condition, en- dometriosis exhibits features reminiscent of malignancy, including abnormal cell migration and invasion. These fac- tors contribute to endometriosis and to its recurrence af- ter surgery. Despite extensive research over the past few decades into the multifactorial causes of endometriosis [10– 13], the underlying mechanism remains unclear. Telomerase plays a vital role in preserving telomere length and ensuring cellular immortality [ 14–16]. With the exception of hematopoietic stem cells and endometrial cells, telomerase is generally not expressed in normal so- matic cells. Nonetheless, telomerase activity is present in various cancers and is linked to their capacity for limit- less replication. Human telomerase reverse transcriptase (hTERT) is a catalytic protein subunit essential for telom- erase function. A significant relationship has been noted be- tween hTERT mRNA levels and telomerase activity across different tissues [ 17,18], with hTERT mRNA serving as a key factor in regulating telomerase activity [ 19]. Telom- erase activity and hTERT mRNA levels in endometrial cancer are significantly higher than in the normal cycling endometrium [ 20]. Interestingly, telomerase activity and hTERT mRNA expression in patients with endometriosis correlate with the proliferative potential of the endometrium [21]. These findings indicate that TERT plays an essential role in the adhesion of endometrial cells, and that abnormal TERT expression may contribute to the pathological pro- cesses involved in endometriosis. However, this connec- tion has yet to be investigated, and the upstream regulatory mechanisms of TERT remain unknown. N6-methyladenosine (m6A) is among the most com- mon modifications found in RNA [ 22,23]. Like DNA methylation, m6A exhibits dynamic and reversible regula- tory properties. This modification is added by a methyl- transferase complex comprising two primary subunits [ 24]. Demethylation of m6A requires the action of fat mass and obesity-associated protein (FTO) [ 25] and alkB ho- molog 5 (ALKBH5) [ 26]. The equilibrium between m6A methyltransferases and demethylases is essential for the dy- namic regulation of m6A. Increasing evidence indicates that m6A plays a significant role in various disease pro- cesses, particularly in cancer metastasis [ 27]. Conversely, reduced methyltransferase-like 14 (METTL14) expression promotes hepatocellular carcinoma cell metastasis by in- fluencing the processing of m6A-dependent primary mi- croRNA (pri-miRNA) [ 28]. However, the precise role of m6A and its associated mechanisms in the development of endometriosis remains elusive and requires further inves- tigation. This study was to explore the influence of m6A modification on the upregulation of TERT expression ob- served in ectopic endometrial tissue. A better understand- ing of the epigenetic regulation of TERT via m6A modifi- cation may shed light on the pathogenesis of endometriosis and identify potential therapeutic targets for this condition. This study provides evidence that reduced m6A mod- ification is a key driving factor for the aberrant expression of TERT in endometriosis. Our findings highlight the sig- nificance of methyltransferase-like 3 (METTL3)/TERT in the development of endometriosis and suggest that TERT could serve as a novel target in the diagnosis and therapy of this condition. 2. Methods 2.1 Patient Enrollment and Tissue Collection Individuals with ovarian endometriosis were chosen from the Obstetrics and Gynecology Department at Li- uzhou Maternal and Child Health Hospital. Post-surgery, the endometrial tissues were categorized into eutopic en- dometrial (EU) and ectopic endometrial (EC). EU samples were sourced from the endometrial lining within the uterus, while EC samples were taken from the ovarian tissue. We included women aged 18–45 with confirmed ovarian en- dometriosis and excluded those with malignancies, autoim- mune diseases, other gynecological conditions, recent hor- mone therapy (within six months), or systemic diseases. To control for age, we focused on women aged 18–45 and ensured all samples were collected during the proliferative phase of the menstrual cycle without recent hormone treat- ment to minimize hormonal and systemic effects. Normal endometrium (normal control; n = 8) was obtained dur- ing the surgical operation. Human tissue samples were obtained with patients’ or their families/legal guardians’ written informed consent, in accordance with the Declara- tion of Helsinki guidelines. The research received approval from the Ethics Committee of Liuzhou Maternal and Child Health Hospital (KS-KY -2020-073). 2.2 Cell Culture Human endometrial stromal cells (HESCs) were sourced from Otwo Biotech (HTX2487-2, Shenzhen, China) and cultured with DMEM as previously [ 29]. The culture medium was enriched with 1.5 g/L sodium bicar- bonate (144-55-8, Sigma-Aldrich, St. Louis, MO, USA), 1% recombinant Human Insulin (ITS)+ Premix (354352, Corning, Corning, NY , USA), 500 ng/mL puromycin, and 10% charcoal-stripped fetal bovine serum (F6765, Sigma- Aldrich), and kept in a humidified incubator at 37 °C in a 5% CO 2 environment. All cell lines were validated by short tandem repeat (STR) profiling and tested negative for mycoplasma. 5-Azacytidine (5-aza-dC) (HY -10586) and Suberoylanilide Hydroxamic Acid (SAHA) (HY -10221) were obtained from MCE (Middlesex County, NJ, USA), while sodium butyrate (NaB) (S1999) was acquired from Selleck. The expression of TERT in HESC cells was as- sessed by real-time quantitative PCR (qPCR) after treat- ment for 12 hours with SAHA (5 µM) or NaB (5 mM), or with 5 µM 5-Aza-dC for 48 hours. 2.3 Lentiviral Transduction To overexpress TERT and METTL3 in HESCs, hu- man TERT and METTL3 were synthesized and cloned into the pLVX plasmid vector (General Biosystems, An- hui, China). For the knockdown of TERT and METTL3, lentiviruses containing small interfering RNAs (siRNAs) targeting TERT (5′-AAAAA TGTGGGGTTCTTCCAA- 3′), METTL3 (5′-TCTAACTCAGGA TCTGTAGCT-3′), or a nonsense siRNA (5 ′-CAGCCA TCAACTCAGA TTGTT- 3′) were constructed by General Biosystems (Anhui, China). Recombinant lentiviral vectors or control vectors, along with two auxiliary packaging plasmids (pSPAX2 and pMD2G), were co-transfected into HEK293T cells to pro- duce the lentiviruses, as provided by General Biosystems 2 (Anhui, China). After 48 hours, the supernatant was col- lected and used to infect HESCs with an initial multiplicity of infection (MOI) of 50. Transduction efficiency was as- sessed 48 hours post-infection using Western blot analysis (Supplementary Fig. 1 ). 2.4 Immunohistochemistry The retrieved tissue specimens were promptly pre- served in 4% buffered formalin and subsequently pre- pared for immunohistochemical (IHC) analysis by Service- bio Biotechnology Co., Ltd. (Wuhan, China). Paraffin- embedded slices were subjected to staining with a primary antibody targeting METTL3 (1:200, Proteintech, 15073-1- AP , San Diego, CA, USA) according to the manufacturer’s instructions. 2.5 RNA m6A Quantitative Assays To assess m6A levels in total RNA samples, an EpiQuik m6A RNA Methylation Quantification Kit (Epi- gentek, P-9005-48, Farmingdale, NY , USA) was utilized. Initially, the RNA was applied to wells pre-coated with RNA high binding solution, then capture and detection an- tibody solutions were introduced. The m6A levels were de- termined by measuring absorbance at 450 nm and calculat- ing relative quantification. 2.6 Cell Proliferation Assays Cell proliferation was evaluated through the Cell Counting Kit-8 (CCK-8) assay (MCE, HY -K0301). Cells were seeded in a 96-well plate and treated as needed. Af- ter treatment, 10 µL of CCK-8 solution was added to each well and incubated at 37 ℃ for 1–4 hours. Absorbance at 450 nm was then measured using a microplate reader to assess cell proliferation and viability. For 5-ethynyl-2’ - deoxyuridine (EdU) staining, the culture medium was sup- plemented with 10 µM EdU reagent (Beyotime Biotechnol- ogy, C0078S, Shanghai, China) and incubated for 3 hours at 37 °C. Cells were then stained with Hoechst 33342 as per the manufacturer’s instructions and examined under a fluorescence microscope. 2.7 Transwell Assays Cell invasion and migration were assessed using Tran- swell chambers (Corning, Corning, NY , USA). For inva- sion assays, Matrigel (Corning, 354248) was mixed with fetal bovine serum (FBS)-free DMEM/F12 medium at a 1:3 ratio and applied to the upper chamber. For migration as- says, Matrigel was not used. Cells (8 × 104) were resus- pended in 200 µL of serum-free DMEM/F12 and placed in the upper chamber, while the lower chamber contained 600 µL of medium with 20% FBS. After 24 hours, cells that ad- hered to the membrane’s underside were fixed, stained, and then quantified using a microscope. 2.8 Wound Healing Assay Once cells reached 90–100% confluence, a sterilized pipette tip (200 µL) was used to create scratches. Following PBS washes to remove detached cells and debris, wound healing was monitored and photographed at 0 and 24 hours post-scratching. The wound area at each time point was compared to the initial size at 0 hours, and the percentage of area closure was calculated. 2.9 RIP Assay The RIP assay was conducted with the Magna™ RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, 17-700, Burlington, MA, USA). Cells were lysed using a RIP lysis buffer, and magnetic beads were conjugated with human antibodies against YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) (Proteintech, 24744-1-AP) or m6A (Synaptic Systems, 202-003, Gottingen, Germany). Normal mouse Immunoglobulin G (IgG) antibody (Milli- pore, 12-370) was used as a negative control. RNA ob- tained with the RIP assay was assessed by RT-qPCR and normalized to the input. 2.10 Western Blotting Total protein was extracted with RIPA buffer (Bey- otime, P0013B). Following protein quantification, SDS- PAGE separation, and transfer onto polyvinylidene diflu- oride (PVDF) membranes were performed. The mem- branes were treated with 5% skim milk to prevent non- specific binding, and then incubated with the specified an- tibodies: TERT (1:1000, Thermo, MA5-16034, Waltham, MA, USA), METTL3 (1:2000, Cell Signaling Technol- ogy, #86132, Danvers, MA, USA.), YTHDF2 (1:2000, Ab- cam, ab246514, Cambridge, UK), YTHDF3 (1:2000, Ab- cam, ab220161), IGF2BP1 (1:2000, Abcam, ab290736), IGF2BP2 (1:2000, Abcam, ab124930), IGF2BP3 (1:2000, Abcam, ab177477), and GAPDH (1:6000, Proteintech, 60004-1-Ig) overnight at 4 °C. Subsequent incubation with secondary antibodies was followed by detection of pro- tein bands using ECL chemiluminescent reagent (Bey- otime, P0018S) and densitometry analysis using ImageJ (version 1.41, LOCI, University of Wisconsin, Madison, WI, USA). 2.11 Luciferase Reporter Assay The luciferase reporter assay was carried out utilizing the Dual-Luciferase Reporter Assay System. Cells were transfected with a pmiGLO-based luciferase vector carry- ing either wild-type or mutated TERT, or a control vec- tor, employing Lipofectamine 3000 reagent (Invitrogen, Waltham, MA, USA). After 24 hours, the luciferase and Renilla activities were measured according to the man- ufacturer’s guidelines. For further analysis, pmirGLO- TERT-3′UTR-WT and pmirGLO-TERT-3 ′UTR-Mut con- structs were introduced into control or shMETTL3 cells for 24 hours, and F-luc and R-luc activities were evaluated. 3 Fig. 1. TERT overexpression in endometriosis. (A,B) Protein (A) and mRNA (B) expression levels of TERT in NC, EU, and EC tissue. (C) Immunohistochemistry of TERT expression in the endometriosis and control groups. Scale bar: 200 µm or 80 µm. Error bars represent the mean ± SD of results from triplicate biological experiments. ** p < 0.01. TERT, telomerase reverse transcriptase; NC, normal endometrial; EU, eutopic endometrial; EC, ectopic endometrial. 2.12 mRNA Stability To measure RNA stability, cells were treated with 5 µg/mL actinomycin D (Act-D, Sigma-Aldric, #A9415). RNA samples were collected at various time intervals post- treatment, and real-time PCR was performed to analyze RNA levels. The TERT mRNA half-life ( t1/2) was cal- culated with the formula ln2/slope, using GAPDH as the

control. 2.13 Real-Time PCR RNA was extracted with Trizol reagent (Invitrogen, A33250). For cDNA synthesis, 500 ng of mRNA was reverse transcribed with the PrimeScript RT Master Kit (Takara, Dalian, China). qPCR was performed using TB Green Premix Ex Taq II (TaKaRa, RR820Q), and transcript levels were determined based on the threshold cycle num- ber. GAPDH served as the normalization control, and rel- ative expression was calculated using the 2 −∆∆Ct method. The primer sequences are listed in Supplementary Table 1. 2.14 Statistical Analysis

are expressed as mean ± SEM. The Shapiro- Wilk test was employed to evaluate data normality. For comparing two groups, an unpaired Student’s t-test was used for normally distributed data, while the Mann- Whitney test was applied for data that did not follow a nor- mal distribution. For multiple group comparisons, ANOV A was performed with subsequent Bonferroni correction to adjust for multiple tests. A significance level of p < 0.05 4 was adopted, and all experiments were performed indepen- dently. Statistical analyses were performed using Graph- Pad Prism software (version 9.1.4, Dotmatics, Boston, MA, USA). 3. Results 3.1 Upregulation of TERT in Ectopic Tissues To explore the involvement of TERT in endometriosis, we first evaluated its expression levels using Western blot- ting and qRT-PCR across normal endometrial (NC), EU, and EC. Our analysis revealed a notable elevation in TERT expression in EC relative to both EU and NC (Fig. 1A,B). No significant variation in expression levels was detected between EU and NC. To confirm these findings, we con- ducted immunohistochemistry (IHC) to assess TERT ex- pression across NC, EU, and EC samples. The IHC results verified higher TERT expression in EC relative to NC and EU (Fig. 1C), supporting the results obtained from qRT- PCR and Western blot analyses. This indicates that in- creased TERT levels may play a role in the progression of endometriosis. 3.2 TERT Promotes the Proliferation and Migration of Endometrial Stromal Cells in Vitro To explore the potential biological function of TERT in HESCs, lentiviruses were used to knockdown (shTERT) or overexpress TERT. EdU assay and CCK-8 assays were then performed to evaluate cell viability following TERT knockdown or overexpression. TERT knockdown signifi- cantly reduced the proliferation of HESCs, whereas TERT overexpression had the opposite effect (Fig. 2A,B). Next, the migratory and invasive abilities of HESCs with altered TERT expression were investigated using wound healing and transwell assays. TERT overexpression was found to promote cell migration and invasion, whereas TERT knockdown inhibited these processes (Fig. 2C,D). These findings indicate that TERT plays a crucial role in promoting cell proliferation and migration during en- dometriosis. 3.3 Downregulation of METTL3-Mediated m6A Modification in Endometriosis Recent studies demonstrated the involvement of epi- genetic mechanisms in gene dysregulation. We therefore investigated whether epigenetic factors contribute to the up- regulation of TERT in endometriosis. TERT expression re- mained unchanged following the treatment of HESCs with a DNA methyltransferase inhibitor (5-aza-dC), suggesting that DNA methylation does not play a role in regulat- ing TERT. Furthermore, the application of broad-spectrum histone deacetylase (HDAC) inhibitors SAHA and NaB did not affect TERT expression, indicating that histone acetylation is not involved in TERT regulation in HESCs (Supplementary Fig. 2 ). m6A modification in endometriosis was investigated by measuring m6A levels in NC, EU, and EC using a col- orimetric method. The results showed that m6A levels were lower in EC compared to NC and EU. No signifi- cant difference in the m6A level was observed between EU and NC (Fig. 3A). The expression of m6A-associated genes in NC, EU, and EC was subsequently evaluated in order to identify the key molecules responsible for reduced m6A in endometriosis. Both the mRNA and protein levels of METTL3 were found to be downregulated in EC rela- tive to NC and EU (Fig. 3B–D). These findings were con- sistent with the lower m6A levels, suggesting a potential role for METTL3 in m6A modification in endometriosis. Moreover, overexpression of METTL3 was found to signif- icantly reduce TERT mRNA and protein expression, while reducing METTL3 levels led to an increase in TERT ex- pression relative to normal controls (Fig. 3E,F). 3.4 Involvement of TERT in METTL3-Induced Cell Proliferation, Migration and Invasion We investigated how TERT influences METTL3- driven cell proliferation, migration, and invasion in HESCs by introducing a METTL3-overexpressing lentivirus into cells with TERT overexpression. It was observed that ele- vated METTL3 levels markedly reduced cell proliferation, migration, and invasion. In contrast, TERT overexpression counteracted these suppressive effects (Fig. 4A–D). These findings indicate that TERT plays a role in modulating the METTL3-mediated inhibition of HESC proliferation, mi- gration, and invasion. 3.5 TERT is Regulated by METTL3-Mediated m6A Modification Emerging evidence suggests that m6A influences var- ious aspects of RNA fate [ 30–32]. We next investigated the role of m6A modification in TERT upregulation. Data from the online RNA modification resource (RMBase, http: //rna.sysu.edu.cn/rmbase/) indicate that multiple m6A sites are present within the TERT transcript ( Supplementary Fig. 3 ). In order to validate whether TERT is a target of METTL3-mediated m6A modification, we performed MeRIP-qPCR in HESCs. A remarkable decrease in m6A modification of TERT was observed when METTL3 was knocked down (Fig. 5A). Furthermore, the distribution of m6A methylation in TERT mRNA was investigated using fragmented RNA isolated from HESCs. The highest level of m6A methylation was observed in the 3 ′ untranslated region (UTR) of TERT (Fig. 5B). Two m6A modification sites were identified within the 3 ′UTR of TERT (Fig. 5C). To investigate the potential role of m6A in the 3 ′UTR, lu- ciferase reporters containing the wild-type or mutant TERT m6A methylation sites were generated (Fig. 5D). A fire- fly luciferase reporter assay demonstrated that, in HESCs with METTL3 knockdown, the luciferase activity of the re- porter with the wild-type site was markedly elevated com- 5 Fig. 2. The impact of TERT on HESC proliferation, migration, and invasion. (A,B) The influence of TERT silencing or overex- pression on HESC proliferation was determined by CCK-8 assay (A) and 5-ethynyl-2’ -deoxyuridine (EdU) incorporation (B) Scale bar: 100 µm. (C) Transwell assays were conducted to evaluate migration (without matrigel) and invasion (with matrigel) 24 hours post-TERT manipulation. Representative images and quantification are displayed. Scale bar: 100 µm. (D) The wound healing assay was employed to measure cell motility at 0 and 24 hours, with representative images and quantitative data provided. Scale bar: 100 µm. Error bars denote the mean ± SD from 3 independent experiments. * p < 0.05, **p < 0.01. HESC, Human endometrial stromal cell; CCK-8, Cell Counting Kit-8; shNC, short hairpin negative control. 6 Fig. 3. METTL3 decreases m6A levels in endometriosis. (A) m6A content in NC, EU, and EC tissue. (B) mRNA expression of m6A- related genes measured by quantitative Reverse Transcription PCR (qRT-PCR). (C) METTL3 protein levels assessed through Western blotting. (D) METTL3 localization in tissues identified via Immunohistochemistry (IHC). Scale bar: 200 µm or 80 µm. (E,F) TERT mRNA levels evaluated by qRT-PCR (E) and Western blotting (F). Error bars indicate the mean± SD from three independent experiments. *p < 0.05, **p < 0.01. METTL3, methyltransferase-like 3; m6A, N6-methyladenosine. 7 Fig. 4. METTL3 regulates HESC proliferation, migration, and invasion via TERT. (A,B) The EdU (A) and CCK-8 assay (B) were used to evaluate the effect of negative control and METTL3-overexpressing vector on HESC proliferation, both in the absence or presence of TERT overexpression. Scale bar: 100 µm. (C) Transwell assays were performed 24 h after transfection. Scale bar: 100 µm. (D) Changes in cell motility were assessed through wound healing assays at 0 and 24 h. Representative images and quantification are presented. Scale bar: 100 µm. Error bars represent the mean ± SD of triplicate biological experiments. ** p < 0.01. 8 pared to that observed in control cells. However, muta- tions in either one or both of the TERT m6A motifs abol- ished the increased luciferase activity observed in cells with METTL3 knockdown (Fig. 5E). These findings suggest that m6A methylation in the TERT 3 ′UTR is responsible for mRNA stability. Additionally, TERT mRNA expression was higher in cells co-transfected with METTL3 and mu- tant TERT-3′UTR sites (TERT-3′UTR-MUTs) compared to cells with the wild-type site (TERT-3 ′UTR-WT) (Fig. 5F). Furthermore, the mRNA stability of TERT-3′UTR-WT was greater in control cells than in cells with METTL3 overex- pression, and the mutation in TERT-3 ′UTR abolished this difference (Fig. 5G,H). These results indicate that m6A in the TERT 3 ′UTR is crucial for mRNA stability, possi- bly due to its impact on the secondary structure of TERT mRNA. 3.6 YTHDF2 Promotes the Degradation of TERT mRNA through an m6A-Dependent Mechanism To regulate gene expression, m6A modification is performed by m6A writers and requires recognition by m6A readers [ 33,34]. m6A readers, such as YTHDF2, YTHDF3, and IGF2BP1~3, can modulate mRNA stabil- ity [ 35,36]. The present study found that YTHDF2, but not YTHDF1, YTHDF3, or IGF2BP1/2/3, showed signif- icant binding to TERT mRNA in HESCs (Fig. 6A). Fur- thermore, the interaction between YTHDF2 and TERT was reduced in HESCs with METTL3 knockdown relative to control cells (Fig. 6B). Stability assays of mRNA indicated that TERT mRNA had an extended half-life in cells with YTHDF2 knockdown, while it was notably shorter in cells with YTHDF2 overexpression (Fig. 6C,D). Consistent with these findings, YTHDF2 negatively regulated TERT ex- pression in HESCs (Fig. 6E,F). Additionally, the expression level of TERT mRNA in cells co-transfected with YTHDF2 and mutant TERT-3′UTR sites (TERT-3′UTR-MUTs) was higher than in cells co-transfected with YTHDF2 and the wild-type site (TERT-3 ′UTR-WT) (Fig. 6G). These findings demonstrate that YTHDF2 recognizes METTL3- methylated TERT mRNA and facilitates its decay (Fig. 7). 4. Discussion Endometriosis is a multifaceted and poorly understood condition that profoundly affects those who suffer from it. Although its origins are thought to be multifactorial, the precise functional and biological processes driving its onset remain largely elusive. V arious studies have exam- ined genes with increased expression in the endometrium of affected individuals. In this study, we explored the in- volvement of human TERT and m6A modification in en- dometriosis to uncover a potential new molecular mecha- nism underlying its development. Human TERT is the catalytic subunit of telomerase and plays a crucial role in its activity [ 37]. The classi- cal functions of TERT are to activate telomerase activity, synthesize telomere DNA, maintain telomere stability, and confer cells with the potential for unlimited proliferation. These functions are closely related to tumorigenesis and development, with TERT being highly expressed in over 90% of human malignancies [ 38]. Besides the telomerase pathway, recent studies have also shown that TERT partic- ipates in the regulation of gene expression and affects tu- mor cell invasion and gene regulation through telomerase- independent mechanisms. Overexpression of TERT re- markably increased the in vitro invasive ability of cells, and that hTERT expression in gastric cancer tissue was closely associated with tumor progression. High expression of TERT was also shown to enhance the invasive ability of tu- mor cells in cervical cancer, osteosarcoma and breast cancer [39]. This was accompanied by upregulation of metallopro- teinase. Although endometriosis is not a malignant disease, many of its clinical and biological manifestations resemble the characteristics of malignant tumors, including abnormal cell proliferation, invasion, diffusion, and even metastasis. Similar to its role in the invasion and metastasis of tumors, hTERT may therefore also have an important role in the in- vasion of ectopic endometrial cells. Our findings revealed that TERT expression was upregulated in ectopic endome- trial (EC) tissue compared to eutopic endometrial (EU) and normal endometrial (NC) tissue. This observation suggests the potential involvement of TERT in the aberrant cellular behaviors associated with endometriosis. To further elu- cidate the function of TERT, we conducted experiments to investigate the impact of its depletion in HESCs. We found that suppression of TERT expression led to a signif- icant decrease in the proliferation, migration, and invasion of HESCs, whereas TERT overexpression had the opposite effect. This result suggests that TERT plays a crucial role in promoting the aggressive behavior of endometrial cells in endometriosis. Epigenetic processes have been linked to gene expres- sion dysregulation across various diseases, including can- cers and developmental disorders [ 40,41]. This study ex- plored whether epigenetic mechanisms contribute to the el- evated expression of TERT seen in endometriosis. We first examined whether DNA methylation and histone acety- lation were involved in TERT expression, but found no significant changes in expression after treatment with in- hibitors targeting these epigenetic modifications. This led us to investigate the potential role of m6A modification, the most frequent RNA modification, in the regulation of TERT expression. Our results showed lower m6A levels in endometriotic tissue compared to NC and EU. Previ- ous research has indicated that decreased expression of the m6A methyltransferase METTL3 facilitates endometriosis development. METTL3 was found to be downregulated in endometriotic stromal cells, which enhances their migration and invasion through the METTL3/m6A/miR126 pathway [42]. Our study further confirmed the reduced expression of METTL3 in endometriotic cells. Additionally, we observed 9 Fig. 5. METTL3-driven m6A modification regulates TERT expression. (A,B) Methylated RNA immunoprecipitation (MeRIP)- qPCR analysis of relative enrichment of m6A on TERT. (C) A diagram illustrates the locations and mutations of m6A sites within the TERT 3 ′UTR, with “mut” denoting mutant and “chr” referring to chromosome. (D) A schematic shows the mutations introduced in the 3 ′UTR to examine the impact of m6A on TERT expression. (E) The relative luciferase activity was measured for in control or shMETTL3 HESCs. (F) TERT-3 ′UTR-WT or TERT-3 ′UTR-DMut was transfected into control or shMETTL3 HESCs for 24 hours, followed by qPCR analysis to determine TERT mRNA expression levels. (G,H) Following 24-hour transfections of TERT-3 ′UTR- WT (G) or TERT-3 ′UTR-DMut (H) into control or shMETTL3 cells, the cells were treated with actinomycin D (5 µg/mL) for various durations. TERT mRNA levels were subsequently measured by qPCR. Error bars indicate the mean ± SD from triplicate experiments. **p < 0.01; ns. indicates not significant. IgG, Immunoglobulin G; WT, wild type. 10 Fig. 6. YTHDF2 regulates the decay of TERT mRNA in an m6A-dependent fashion. (A) TERT mRNA was examined using RIP- qPCR with antibodies specific to YTHDF2, YTHDF3, and IGF2BP1~3. (B) The binding of YTHDF2 to TERT mRNA was assessed in control and shMETTL3 cells via RIP-qPCR. (C,D) The degradation rate of TERT mRNA was analyzed at various time points after treating with actinomycin D (5 µg/mL). (E,F) The levels of TERT protein (E) and TERT mRNA (F) were measured following the suppression or overexpression of YTHDF2 in HESCs. (G) TERT-3 ′UTR-WT or TERT-3 ′UTR-DMut was transfected into control or YTHDF2-overexpressing HESCs for 24 hours, after which TERT mRNA expression was quantified using qPCR. Error bars denote the mean ± SD from triplicate biological experiments. ** p < 0.01. YTHDF2, YTH N6-methyladenosine RNA binding protein 2. 11 Fig. 7. Graphical representation of the roles of METTL3 and TERT in endometriosis. METTL3 modifies TERT mRNA through m6A, while YTHDF2 mediates the decay of TERT mRNA in an m6A-dependent manner. that overexpression of METTL3 led to decreased TERT ex- pression, while METTL3 knockdown resulted in increased TERT levels. These results imply that METTL3-mediated m6A modification plays a role in regulating TERT expres- sion in endometriosis. Moreover, the impact of METTL3- mediated m6A modification on TERT mRNA stability ap- pears to be YTHDF2-dependent, suggesting this regulatory axis is involved in the pathogenesis of endometriosis. Our study highlights a novel molecular mechanism in- volving METTL3-mediated m6A modification of TERT, which could have significant clinical implications. By elu- cidating the role of TERT and METTL3 in endometriosis, our findings provide a foundation for developing targeted therapies aimed at modulating this pathway. Inhibiting METTL3 or altering m6A modification could potentially offer new treatment options for managing endometriosis, improving patient outcomes, and enhancing quality of life. However, translating these insights into clinical practice will require additional research and validation in clinical settings. Despite the valuable insights provided by our study, several limitations must be acknowledged. Our findings are primarily based on in vitro experiments using HESCs and tissue samples. The absence of in vivo ani- mal model studies limits our ability to fully understand how TERT and METTL3-mediated m6A modification impact endometriosis in a living organism. Further research us- ing animal models is needed to validate these findings and assess their relevance to human disease. While our study identifies a potential role of METTL3-mediated m6A mod- ification in regulating TERT mRNA stability and HESC cell behavior, the precise mechanisms underlying these interac- tions remain to be fully elucidated. Additional research is needed to explore how METTL3 influences TERT function and to identify other potential regulatory factors involved in this pathway. 12 5. Conclusions Our study reveals a new molecular mechanism in en- dometriosis, suggesting that dysregulation of m6A modifi- cation through the METTL3/TERT axis enhances the mi- gration and invasion of endometriotic cells. This pathway may offer potential therapeutic targets for novel treatments. However, the study’s limitations include its reliance on in vitro cell lines, necessitating further validation in animal models. Additionally, the interaction between TERT and m6A modification in endometriosis needs further investi- gation. In summary, our findings highlight the role of the METTL3/TERT axis in endometriosis and pave the way for future research and targeted therapies. Availability of Data and Materials The datasets used and/or analyzed during the current study are available from the corresponding author on rea- sonable request. Author Contributions Conceptualization: JJC, YZ and FL; data acquisition: HT, YNW, RM, YSL, LFN and FL; data analysis: JJC, YZ, HT, YNW and FL; original draft preparation: JJC, YZ, HT, YNW and FL; review and editing: JJC, YZ, HT, YNW, RM, YSL, LFN and FL; all authors revised and agreed the final version of the manuscript. All authors have partici- pated sufficiently in the work and agreed to be accountable for all aspects of the work. Ethics Approval and Consent to Participate Human tissue samples were obtained with patients’ or their families/legal guardians’ written informed consent, in accordance with the Declaration of Helsinki guidelines. The research received approval from the Ethics Committee of Liuzhou Maternal and Child Health Hospital (KS-KY - 2020-073). Acknowledgment We would like to express our sincere gratitude to all individuals and organizations who have contributed to this research. Special thanks to Y anjun Zheng for their invalu- able guidance and support throughout this study. Funding This work was funded by the Guangxi Science and Technology Plan Project (Guangxi Clinical Research Center for Obstetrics and Gynecology), grant #GuiKe AD22035223, and Liuzhou Science and Technology Plan Project, grant #2020NBAB0825. Also was supported by Foundation of State Key Laboratory of Ultrasound in Medicine and Engineering [Grant No.2021KFKT020]. Conflict of Interest The authors declare no conflict of interest. Supplementary Material Supplementary material associated with this article can be found, in the online version, at https://doi.org/10. 31083/j.fbl2912421.

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