Leuprorelin Suppresses Angiogenesis and Malignant Progression in Endometrial Cancer via Downregulation of the ELAVL1/VEGFA Axis.

NingNing Zhang: conceptualization, investigation, writing – original draft, writing – review and editing. Fang Zhao: data curation, formal analysis, methodology, writing – original draft. LiMei Tan: data curation, formal analysis, investigation, methodology.

All animal experiments complied with the ARRIVE guidelines and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The experiments were approved by the Institutional Animal Care and Use Committee of Shijiazhuang People's Hospital.

The authors have nothing to report.

Leuprorelin is principally employed clinically for management of hormone‐related disorders. Given the established association of EC with estrogen dysregulation, this study probed its molecular mechanism of action in EC pathophysiology. CCK‑8 assays were first used to evaluate the effects of leuprorelin (0.5–8 μM) on the viability of HESC and HEC‑1 A cells (Figure  1A ). Results showed that after 48 h, leuprorelin inhibited HEC‑1 A cells more strongly than HESC cells in a dose‑dependent manner, suggesting greater sensitivity of cancer cells. Following CCK‐8 analysis, 2 μM leuprorelin was selected as the working concentration for downstream phenotypic assays in HEC‐1A cells. Colony formation assays revealed significantly reduced colony numbers in leuprorelin‑treated cells compared with controls (Figure  1B ). EdU incorporation assays confirmed that leuprorelin inhibited cell proliferation (Figure  1C ). Transwell assays showed that leuprorelin reduced the number of migrated cells compared with controls (Figure  1D ). Wound‑healing assays revealed slower wound closure in leuprorelin‑treated cells than in controls (Figure  1E ). Flow cytometry revealed that leuprorelin significantly increased the apoptosis rate in HEC‑1 A cells (Figure  1F ). Leuprorelin Significantly Suppresses Malignant Phenotypes of EC Cells. (A) The inhibitory rates of HESC and HEC‐1A cells treated with leuprorelin were assessed by CCK‐8 assay. (B, C) Proliferation of HEC‐1A cells with or without leuprorelin treatment was determined by colony formation assay (B) and EdU assay (C). Scale bar, 100 μm. (D) Migration of HEC‐1A cells with or without leuprorelin treatment was evaluated using Transwell assay. Scale bar, 100 μm. (E) Migration of HEC‐1A cells with or without leuprorelin treatment was assessed by wound healing assay. (F) Apoptosis of HEC‐1A cells with or without leuprorelin treatment was analyzed by flow cytometry. Error bars represent the mean ± SD of at least three independent experiments. * p  < 0.05, ** p  < 0.01, *** p  < 0.001, **** p  < 0.0001. Statistical significance was calculated using two‐tailed unpaired Student's t ‐test (for two groups) or one‐way ANOVA followed by Tukey's test (for more than two groups). ELAVL1 has been shown to promote EC progression in previous studies [ 16 ]. Analysis of TCGA‑UCEC data revealed significantly higher ELAVL1 expression in EC tissues than in normal endometrium (Figure  2A ). In addition, previous studies have confirmed that ELAVL1 participates in GnRHR activation‑mediated gene regulation [ 17 ]; accordingly, ELAVL1 expression following leuprorelin treatment was further investigated. RT‐qPCR and Western blot analyses demonstrated significantly decreased ELAVL1 mRNA and protein levels in HEC‐1A cells following leuprorelin treatment (Figure  2B,C ). VEGFA, a well‑established target of ELAVL1 [ 18 , 19 ], positively correlated with ELAVL1 expression in EC samples (R = 0.43, p  < 0.05) (Figure  2D ). Leuprorelin also decreased VEGFA mRNA and protein in HEC‑1 A cells (Figure  2E,F ). VEGFA is a key regulatory gene in angiogenesis signaling pathways (Figure  2G ). Therefore, angiogenesis in HUVECs was examined following leuprorelin treatment to assess functional consequences of VEGFA downregulation. Tube formation assays showed that leuprorelin significantly reduced angiogenic capacity (Figure  2H ). Leuprorelin Suppresses the Expression of ELAVL1 and VEGFA. (A) Differential expression analysis of ELAVL1 was performed using TCGA‐UCEC data. (B, C) ELAVL1 mRNA (B) and protein (C) levels in HEC‐1A cells after leuprorelin treatment were analyzed by RT‐qPCR and Western blot. (D) Correlation analysis between ELAVL1 and VEGFA expression was performed using TCGA‐UCEC data. (E, F) VEGFA mRNA (E) and protein (F) levels in HEC‐1A cells after leuprorelin treatment were analyzed by RT‐qPCR and Western blot. (G) Pathway enrichment analysis for VEGFA was performed using TCGA‐UCEC data. (H) Angiogenesis was examined by tube formation assay in HUVECs following leuprorelin treatment. Error bars represent the mean ± SD of at least three independent experiments. * p  < 0.05, ** p  < 0.01, *** p  < 0.001, **** p  < 0.0001. Statistical significance was calculated using two‐tailed unpaired Student's t ‐test (for two groups) or one‐way ANOVA followed by Tukey's test (for more than two groups). The regulatory mechanism through which ELAVL1 modulates VEGFA expression was subsequently investigated in detail. ELAVL1 expression was first examined across a panel of EC cell lines (HEC‐1A, HEC‐1B, RL95‐2, KLE) and human normal endometrial stromal cell line HESC (Figure  3A ). Based on differential expression results, HEC‐1A cells were selected for ELAVL1 knockdown experiments, while KLE cells were selected for ELAVL1 overexpression studies. RT‑qPCR and Western blot analyses confirmed the knockdown and overexpression efficiencies (Figure  3B,C ). Subsequently, VEGFA mRNA and protein expression were detected following ELAVL1 knockdown or overexpression manipulations. Results showed significantly decreased VEGFA expression following ELAVL1 knockdown and significantly increased expression following ELAVL1 overexpression (Figure  3D,E ). Actinomycin D chase assays showed that ELAVL1 knockdown shortened the half‑life of VEGFA mRNA, whereas overexpression extended it (Figure  3F,G ). RNA‑FISH combined with immunofluorescence showed cytoplasmic colocalization of ELAVL1 and VEGFA in KLE cells (Figure  3H ). RIP‑PCR confirmed direct binding between ELAVL1 and VEGFA mRNA (Figure  3I ). These collective experimental findings indicate that ELAVL1 directly binds to and stabilizes VEGFA mRNA. ELAVL1 Directly Binds to and Stabilizes VEGFA mRNA. (A) ELAVL1 expression was detected by RT‐qPCR and Western blot in EC cell lines (HEC‐1A, HEC‐1B, RL95‐2, KLE) and the human normal endometrial stromal cell line HESC. (B, C) Knockdown (B) and overexpression (C) efficiencies of ELAVL1 in EC cells were verified by RT‐qPCR and Western blot. (D‐E) VEGFA expression was detected by RT‐qPCR and Western blot following ELAVL1 knockdown (D) or overexpression (E). (F, G) Remaining VEGFA mRNA was detected by RT‐qPCR in cells with ELAVL1 downregulation (F) or upregulation (G) after ActD treatment. (H) Colocalization of ELAVL1 and VEGFA in KLE cells was visualized by RNA‐FISH coupled with immunofluorescence. (I) The direct binding of ELAVL1 to VEGFA mRNA was detected by RIP‐PCR. Error bars represent the mean ± SD of at least three independent experiments. * p  < 0.05, ** p  < 0.01, *** p  < 0.001, **** p  < 0.0001. Statistical significance was calculated using two‐tailed unpaired Student's t ‐test (for two groups) or one‐way ANOVA followed by Tukey's test (for more than two groups). To determine whether VEGFA is required for leuprorelin's effects, we established KLE cells with stable VEGFA overexpression, confirmed at mRNA and protein levels (Figure  4A ). Phenotypic changes in cells following leuprorelin treatment were examined across multiple functional assays. Under leuprorelin stimulation conditions, colony formation and EdU incorporation assays demonstrated that VEGFA‐overexpressing cells proliferated more rapidly compared with control groups (Figure  4B,C ). Transwell migration and wound healing assays revealed significantly increased migratory capacity in VEGFA‐overexpressing cells compared with controls (Figure  4D,E ). Flow cytometric apoptosis analysis demonstrated significantly reduced apoptosis rates following VEGFA overexpression (Figure  4F ). Tube formation assay results showed increased angiogenic capacity of HUVECs cultured with conditioned medium from VEGFA‐overexpressing cells compared with controls (Figure  4G ). VEGFA Overexpression Reverses the Antitumor Effects of Leuprorelin. (A) Overexpression efficiency of VEGFA in KLE cells was confirmed by RT‐qPCR and Western blot. (B, C) Proliferation of cells overexpressing VEGFA following leuprorelin treatment was assessed by colony formation (B) and EdU (C) assays. Scale bar, 100 μm. (D) Migration of cells overexpressing VEGFA following leuprorelin treatment was evaluated using Transwell assay. Scale bar, 100 μm. (E) Migration of cells overexpressing VEGFA following leuprorelin treatment was assessed by wound healing assay. (F) Apoptosis of cells overexpressing VEGFA following leuprorelin treatment was analyzed by flow cytometry. (G) Angiogenesis of HUVECs co‐cultured with cells overexpressing VEGFA following leuprorelin treatment was evaluated by tube formation assay. Error bars represent the mean ± SD of at least three independent experiments. * p  < 0.05, ** p  < 0.01, *** p  < 0.001, **** p  < 0.0001. Statistical significance was calculated using two‐tailed unpaired Student's t ‐test (for two groups) or one‐way ANOVA followed by Tukey's test (for more than two groups). Based on in vitro experimental results, a nude mouse xenograft model of EC was established to validate findings in vivo. Compared with control groups, tumor volume and weight were significantly reduced in leuprorelin‐treated groups, while tumor volume and weight were restored to near‐control levels in VEGFA‐overexpressing groups (Figure  5A–C ). Immunohistochemical analysis showed decreased positive staining areas for ELAVL1 and VEGFA in tumor tissues from leuprorelin‐treated groups (Figure  5D ), and reduced microvessel density as assessed by CD31 staining in tumor tissues from treatment groups (Figure  5E ). Additionally, Ki67 proliferation marker expression and TUNEL‐positive apoptotic cells were examined in control and treatment groups. Results demonstrated that following leuprorelin treatment, Ki67 fluorescence intensity decreased while TUNEL‐positive apoptotic rates significantly increased (Figure  5F,G ). Leuprorelin Inhibits Tumor Growth and Angiogenesis in vivo by Suppressing the ELAVL1/VEGFA Axis. (A) Representative images of xenograft tumors in nude mice from each group ( n  = 5 per group). (B) Tumor growth curves showing volume (calculated as 0.5 × length × width 2 ) measured weekly for four weeks. (C) Weights of excised xenograft tumors. (D) Immunohistochemical staining detecting ELAVL1 and VEGFA expression in tumor tissues from mice in the indicated groups. (E) Immunohistochemical staining detecting microvessel density (CD31) in tumor tissues. (F) Immunofluorescence staining detecting Ki67 expression in xenograft tumor tissues. Scale bar, 100 μm. (G) TUNEL staining detecting apoptotic cells in tumor tissues. Scale bar, 100 μm. Error bars represent the mean ± SD of at least three independent experiments. * p  < 0.05, ** p  < 0.01, *** p  < 0.001, **** p  < 0.0001. Statistical significance was calculated using two‐tailed unpaired Student's t ‐test (for two groups) or one‐way ANOVA followed by Tukey's test (for more than two groups).

This study provides the first experimental evidence that leuprorelin, a GnRH agonist, suppresses malignant progression in EC by modulating a post‐transcriptional regulatory axis. Specifically, leuprorelin downregulates ELAVL1, thereby destabilizing VEGFA mRNA and reducing VEGFA protein. These findings expand the understanding of leuprorelin's direct anti‐neoplastic mechanisms beyond its classical role in hormonal suppression and highlight a novel avenue for developing targeted EC therapies. Traditionally, as a GnRH receptor agonist, leuprorelin inhibits estrogen synthesis by downregulating the pituitary‐gonadal axis, thus limiting the growth of estrogen‐dependent (Type I) EC [ 20 , 21 ]. However, current data reveal that leuprorelin significantly inhibits proliferation and invasion even in estrogen receptor‐negative cell lines (e.g., HEC‐1A). This suggests the activation of estrogen‐independent anticancer pathways that operate alongside hormonal effects. Accumulating evidence indicates that leuprorelin's antitumor effects are not confined to hormone‑dependent cancers [ 22 ]; it also shows activity in hormone‑independent tumors such as castration‑resistant prostate cancer [ 23 ], triple‑negative breast cancer [ 24 ], nasopharyngeal carcinoma [ 17 ], and melanoma [ 25 ]. These tumors often express ectopic GnRH receptors, providing a basis for direct antitumor actions that are independent of circulating hormone levels. Currently identified non‑hormonal mechanisms include: (1) inhibition of proliferation— leuprorelin binding to tumor GnRHR triggers calcium signaling and reduces viability [ 17 ]; (2) inhibition of metastasis—leuprorelin downregulates α3 integrin and MMP2, thereby decreasing metastatic potential [ 26 ]. In this study, leuprorelin treatment markedly reduced ELAVL1 protein levels. This is consistent with earlier work linking ELAVL1 to GnRH receptor‑mediated gene regulation and LH secretion, and suggesting that other trans‑acting factors may also affect mRNA fate during GnRH stimulation [ 25 ]. RNA‑binding proteins are key trans‑acting factors that bind cis‑elements in target mRNAs, modulating stability and translation. Their regulatory networks can be double‑edged in disease [ 27 ]. As a central regulator of mRNA stability, ELAVL1, when suppressed, triggers degradation of pro‑oncogenic transcripts, leading to tumor inhibition. In gastric cancer, ELAVL1 promotes progression and metastasis by stabilizing TL1A mRNA [ 28 ]. In breast cancer, ELAVL1 facilitates cell migration and invasion via SOX9 mRNA stabilization [ 29 ]. Beyond stabilizing transcripts, ELAVL1 acts as an oncogene that drives proliferation, metastatic colonization, and chemoresistance [ 30 , 31 , 32 ]. In lung cancer, ELAVL1 is regulated by long non‑coding RNAs, enhancing proliferation, invasion, and 5‑fluorouracil resistance via PI3K/Akt [ 33 ]. Epigenetic modifications of ELAVL1, including N 6 ‑methyladenosine methylation, are implicated in hepatocellular carcinoma development [ 34 ]. In colorectal cancer, ELAVL1 synergizes with 5‑methylcytosine recognition to promote tumorigenesis, highlighting its dual potential as a therapeutic target and prognostic marker [ 35 ]. Collectively, these studies confirm that ELAVL1, as a pivotal RNA‐binding protein, plays critical roles across various malignancies, offering diverse strategies for therapeutic intervention. In this study, VEGFA was identified as a key downstream effector mediating the antitumor activity of leuprorelin via the ELAVL1 axis. Under normal conditions, ELAVL1 stabilizes VEGFA mRNA, elevating VEGFA expression to promote angiogenesis and malignant phenotypes; leuprorelin treatment effectively interrupts this process. VEGFA is a master regulator of angiogenesis in both physiological and pathological settings [ 36 ]. Similar regulatory circuits have been reported: miR‑205‑5p inhibits angiogenesis by downregulating VEGFA and FGF1 in gastric cancer [ 37 ]; CTEN suppresses VEGFA via β‑catenin in breast cancer [ 38 ]; and miR‑203 targets VEGFA in cervical cancer [ 39 ]. In non‑small cell lung cancer, BMI1 promotes H2A ubiquitination, which reduces ZNF24 and enhances VEGFA secretion, thereby supporting angiogenesis [ 40 ]. Anti‑VEGFA therapies, combined with surgery and chemotherapy, have shown promising clinical results [ 41 ]. Identification of angiogenic biomarkers remains important for improving survival and refining prognosis [ 42 , 43 ]. Thus, leuprorelin‑mediated suppression of VEGFA and angiogenesis represents a significant therapeutic mechanism in EC. Several limitations should be acknowledged. The upstream signaling pathways by which leuprorelin regulates ELAVL1 expression and localization remain to be elucidated. Additionally, VEGFA is only one of many ELAVL1 targets; other downstream factors may also affect tumor progression and resistance.

This study elucidates a hormone‑independent mechanism by which leuprorelin inhibits EC progression: it downregulates ELAVL1, thereby destabilizing VEGFA mRNA, a key angiogenic factor. These findings offer new insights into targeted therapy for EC and suggest that the ELAVL1/VEGFA axis may serve as a prognostic biomarker and a target for combination strategies.

Globally, endometrial cancer (EC) is one of the three most common gynecological malignancies, and its incidence has been rising over recent decades, predominantly affecting perimenopausal and postmenopausal women [ 1 ]. Although early diagnosis is generally associated with favorable outcomes, patients with advanced‐stage or recurrent disease have a 5‐year survival rate below 20%. Prolonged exposure to unopposed estrogen remains the primary driver of endometrial carcinogenesis [ 2 ]. Although surgical resection serves as the cornerstone of management [ 3 ], often combined with radiotherapy, systemic chemotherapy, or hormonal interventions [ 4 ], resistance to these modalities remains a major clinical challenge. Recent advancements in EC treatment increasingly underscore the necessity of genomic profiling and molecularly targeted strategies [ 5 ]. Therefore, deciphering the molecular networks governing therapeutic responses in EC is essential for devising rational treatment protocols and improving clinical outcomes. Leuprorelin, a gonadotropin‐releasing hormone (GnRH) agonist, inhibits estrogen biosynthesis by desensitizing pituitary gonadotropin secretion. This mechanism underpins its widespread clinical use in hormone‐dependent disorders such as advanced prostate cancer, endometriosis, and central precocious puberty [ 6 ]. In addition to these conventional uses, leuprorelin is used in combination with the oral GnRH antagonist relugolix for uterine leiomyoma management [ 7 ], while prolonged treatment has been shown to be effective in precocious puberty cases complicated by osteoporosis [ 8 ]. Emerging evidence suggests that leuprorelin may also possess direct antineoplastic properties in peripheral tissues [ 9 ]. Despite its limited success as a monotherapy for advanced or recurrent EC [ 10 ], accumulating evidence suggests its potential value within broader EC treatment strategies [ 11 ]. However, the specific molecular pathways mediating leuprorelin's effects in EC pathophysiology remain incompletely understood and warrant further exploration. ELAVL1 (also known as HuR) is an RNA‐binding protein that stabilizes mRNA by binding to AU‐rich elements (AREs) in their 3′‐untranslated regions. Overexpression of this protein has been observed across a wide spectrum of cancers. In nasopharyngeal carcinoma, ELAVL1 promotes glycolysis via the HMGB3/β‐catenin signaling axis [ 12 ]. In gliomas, it facilitates cell fusion and the formation of tunneling nanotubes, mechanisms that are critical for maintaining tumor heterogeneity [ 13 ]. Additionally, ELAVL1 participates in N 6 ‐methyladenosine (m 6 A)‐dependent transcript regulation [ 14 , 15 ] and influences tumor immune responses. Within the context of EC, ELAVL1 has been shown to regulate Beclin1 expression through the lncRNA NEAT1 axis, thereby modulating cell proliferation, invasion, and migration [ 16 ]. Previous studies have confirmed that ELAVL1 participates in gene regulation mediated by GnRH receptor (GnRHR) activation [ 17 ], suggesting that it may serve as a key downstream molecule for the non‑hormonal antitumor effects of leuprorelin. Therefore, the present study aimed to validate the inhibitory effects of leuprorelin on endometrial cancer cells and to investigate whether these effects are mediated by modulation of ELAVL1.

The authors declare no conflicts of interest.

The human EC cell lines HEC‐1A, HEC‐1B, RL95‐2, and KLE, as well as the normal endometrial stromal cell line HESC and human umbilical vein endothelial cells (HUVECs), were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HEC‐1A cells were maintained in McCoy's 5 A medium, and HEC‐1B cells in Minimum Essential Medium (both from Gibco, Grand Island, NY, USA). KLE and RL95‐2 cells were cultured in DMEM/F12 (Gibco), and HESCs in Dulbecco's Modified Eagle Medium (DMEM). All media were supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin‐streptomycin solution (Gibco). Cells were maintained at 37°C in a humidified incubator with 5% CO 2 atmosphere. For functional experimentation, cells were exposed to escalating concentrations of leuprorelin (0.5–8 μM; Livzon, Zhuhai, China) for a duration of 48 h. Short hairpin RNA targeting ELAVL1 (sh‐ELAVL1) and negative control shRNA (sh‐NC) were designed and synthesized by GeneSeed Biotechnology (Guangzhou, China). Overexpression plasmid vectors pcDNA3.1‐ELAVL1 and pcDNA3.1‐VEGFA were provided by GenePharma (Shanghai, China), with empty pcDNA3.1‐NC vector used as control. HEC‐1A or KLE cells were transfected with these plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Total RNA and protein were harvested 48 h following transfection procedures. Proteins were extracted with RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with protease and phosphatase inhibitors. Protein concentrations in clarified lysates were determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, MA, USA). Aliquots (15 μg protein) were resolved by 10% SDS‐polyacrylamide gel electrophoresis (BioRad, Berkeley, CA, USA) and subsequently electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA). After blocking with 5% skim milk for 1 h at room temperature, membranes were incubated overnight at 4°C with primary antibodies targeting ELAVL1 (1:500, Abcam, Cambridge, UK, Cat# ab200342), VEGFA (1:500, Abcam, Cat# ab46154), and β‐actin (1:1000, Abcam, Cat# ab8226). After extensive washing procedures, membranes were incubated with horseradish peroxidase‐conjugated goat anti‐rabbit IgG secondary antibody (1:1000, VSA27, VICMED, Xuzhou, China) for 1 h at room temperature. Immunoreactive bands were visualized using an enhanced chemiluminescence (ECL) detection system (Thermo Fisher Scientific) and imaged with a gel documentation system (UVP, Upland, CA, USA). Total RNA was extracted from EC cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Following the manufacturer's guidelines, mRNA was reverse‐transcribed into cDNA utilizing the M‐MLV RTase cDNA Synthesis Kit (Takara, Dalian, China). RT‐qPCR was performed on an ABI PRISM 7700 instrument (Thermo Fisher Scientific) utilizing SYBR GreenER qPCR SuperMix (Thermo Fisher Scientific). PCR primers were designed by Invitrogen and Takara and are enumerated in Table  1 . The RT‐qPCR reaction mixture (25 μl) contained cDNA (0.2 μg), primers (200 nM each), and SuperMix components. Amplification conditions comprised initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 15 s and annealing/extension at 60°C for 45 s. Relative gene expression was quantified employing the 2 −ΔΔCt method, normalizing to β‐actin as the endogenous control using ABI Prism 7700 SDS Software (Thermo Fisher Scientific); primer details are provided in Table  1 . Primer Sequences for RT‐qPCR. To inhibit ongoing transcriptional activity, EC cells were treated with 5 μg/mL actinomycin D (Sigma‐Aldrich, St. Louis, MO, USA). Cellular samples were collected at designated time points including 0, 4, 8, 12, and 24 h after treatment. Subsequently, total RNA was extracted from each time point using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to manufacturer specifications. Cells were lysed in RIP lysis buffer (Beyotime) containing protease and RNase inhibitors. Lysates were immunoprecipitated with anti‑ELAVL1 antibody or control IgG for 4 h, followed by incubation with protein A/G magnetic beads (MCE, Shanghai, China). After extensive washing, the bound RNA was isolated using TRIzol reagent (Invitrogen) in accordance with the manufacturer's protocol. Purified RNA was then reverse‐transcribed into cDNA employing the PrimeScript RT Reagent Kit with gDNA Eraser (Takara). Finally, the enriched transcripts were analyzed via RT‐qPCR using SYBR Premix ExTaq (Takara), or alternatively, semi‐quantitative assessment was performed by direct RIP‐PCR. This assay was performed using a FISH kit (GenePharma) according to the manufacturer's protocol. Initially, cells were fixed with 4% paraformaldehyde for 10 min, followed by permeabilization using phosphate‐buffered saline supplemented with 0.5% Triton X‐100 at 4°C for 5 min. After blocking procedures, samples were incubated overnight at 4°C in darkness with primary antibody against ELAVL1 (dilution 1:500; ab200342, Abcam). On the following day, cells were rinsed three times with PBS for 5 min per wash. Next, specimens were incubated with Alexa Fluor 488‐conjugated goat anti‐rabbit IgG secondary antibody (1:500; ab236553, Abcam) for 1 h at room temperature protected from light, followed by three additional PBS washes of 5 min each. RNA FISH procedures were then initiated either concurrently with or immediately following the immunofluorescence washing steps. Specifically, 200 μL of pre‐hybridization buffer was applied to each well for 30 min. Meanwhile, the Cy3‐labeled VEGFA probe (2.5 μL, 20 μmol/L; GenePharma) was pre‐warmed in hybridization solution at 37°C in darkness. Subsequently, 100 μL of the probe‐containing hybridization mixture was added to cells for overnight incubation at 37°C in darkness. Following removal of excess liquid, nuclei were counterstained with DAPI solution for 15 min. Finally, slides were mounted and imaged using fluorescence microscopy. EC cells were seeded into 24‐well plates and maintained under standard culture conditions for 48 h. Cells were incubated with 200 μL fresh medium containing 50 μmol/L EdU (Beyotime) for 2 h. Subsequently, the cells were washed with phosphate‐buffered saline (PBS; Thermo Fisher Scientific) and fixed with paraformaldehyde. To quench free aldehyde groups, cells were treated with 200 μL of glycine solution (2 mg/mL) and rinsed again with PBS. Permeabilization was performed using 0.5% Triton X‐100, after which the cells were incubated with Apollo 488 fluorescent dye for EdU detection. Nuclear counterstaining was conducted using DAPI. Fluorescence images were acquired using an Olympus FSX 100 microscope (Tokyo, Japan), and the proportion of EdU‐positive cells was quantified using ImageJ software. To assess clonogenic potential, log‑phase EC cells were harvested and resuspended as single cells. Following cell counting, equal numbers of viable cells were plated into 6‐well plates at a low density (500–1000 cells/well) and incubated for 14 days to enable colony formation. Upon completion of the culture period, colonies were fixed by adding 2 mL of methanol to each well and incubated at room temperature for 15 min. The fixed cells were then stained with Giemsa solution for 9 min. Finally, images of the stained colonies were captured using a digital camera, and the colonies were counted for statistical analysis. Migration was assessed using Transwell chambers (8 μm pore size). Cells (3 × 10 4 /mL) in serum‑free medium were placed in the upper chambers, while the lower chambers contained medium with 15% FBS as a chemoattractant. Following 48 h incubation at 37°C, non‐migrated cells remaining on the upper membrane surface were gently removed using cotton swabs. Cells that had successfully migrated to the lower membrane surface were subsequently fixed with methanol for 10 min, stained with Giemsa solution for 7 min, and rinsed twice with PBS. After air‐drying, the membranes were carefully excised, mounted on glass slides using neutral balsam, and visualized under a microscope for cell quantification across multiple fields. Cells were seeded in 6‑well plates at 1 × 10 6 /well. When monolayers reached approximately 90% confluence, medium was replaced with serum‑free medium and cells were incubated overnight to minimize proliferation artifacts. Linear wounds were then created by scraping the cell layer with sterile 200 μL pipette tips. Phase‐contrast images were acquired at 0 and 24 h using an Olympus IX53 microscope (Olympus). The wound areas were quantified using LCmicro software (Olympus Soft Imaging Solutions GmbH), and the results are expressed as the percentage of the remaining gap area at 24 h relative to the initial area at 0 h. TUNEL staining was performed using a cell death detection kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, following deparaffinization and rehydration procedures, 5 μm paraffin‐embedded tissue sections were permeabilized with 0.1% Triton X‐100 for 8 min. Subsequently, cells and tissue sections were incubated with TUNEL reaction mixture at 37°C for 1 h. Nuclei were stained with DAPI‐containing mounting medium. TUNEL‐positive cells were observed under a fluorescence microscope (Nikon, Tokyo, Japan). Apoptotic index was calculated as the ratio of TUNEL‐positive cells to total DAPI‐positive nuclei within identical areas from five randomly selected microscopic fields per sample. To assess apoptosis, stably transfected cells were cultured for 48 h, harvested, and washed with PBS. Apoptosis was determined using an Annexin V FITC/PI kit (Keygen Biotech, Nanjing, China) and analyzed on a CytoFLEX flow cytometer (Beckman, Brea, CA, USA). HUVECs were maintained in DMEM with 10% FBS and 1% penicillin‑streptomycin. For tube formation, 1–2 × 10 4 cells were seeded into 96‑well plates pre‑coated with 50 μL growth factor‑reduced Matrigel. Cells were treated with conditioned medium containing 2 μM leuprorelin or vehicle control. For tube formation observation and quantification, cells were cultured under standard conditions for 24 h. Five random microscopic fields were selected and examined using Chemi Imager 5500 V2.03 software (Alpha Innotech, San Leandro, CA, USA) for quantification of tubular structures. All animal procedures strictly adhered to the National Act on the Use of Laboratory Animals (P.R. China) and received formal approval from the Animal Ethics Committee of Shijiazhuang People's Hospital. Four‑ to six‑week‑old female BALB/c nude mice were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China) and maintained under specific pathogen‐free (SPF) conditions. Mice were randomly assigned to three groups ( n  = 5 per group): control, leuprorelin‐treated (5 mg/kg, i.p., twice weekly), and VEGFA‐overexpressing KLE cell group (inoculated subcutaneously with 5 × 10 6 cells in 100 μL PBS). Tumor establishment was achieved by subcutaneously injecting the cell suspension into the right flank of each mouse. Four weeks post‐inoculation, the animals were sacrificed via cervical dislocation, and the resulting tumors were excised for further evaluation. Each tumor was photographed and weighed, while its volume (mm 3 ) was determined using the standard equation: 0.5 × length × width 2 . Xenograft tumors were fixed in 4% paraformaldehyde, dehydrated in graded ethanol, embedded in paraffin, and sectioned at 5 μm. Standard IHC was performed. Deparaffinized slides were subjected to antigen retrieval using either 10 mM sodium citrate buffer (pH 8.0) or Tris‐EDTA buffer (pH 9.0) in a pressure cooker. To suppress endogenous peroxidase activity and minimize non‐specific binding, sections were treated with 3% hydrogen peroxide followed by normal serum blocking. Subsequently, primary antibodies targeting ELAVL1 (1:500; ab200342, Abcam), VEGFA (1:500; ab46154, Abcam), and CD31 (1:500; ab28364, Abcam) were applied and incubated overnight at 4°C in a humidified chamber. Detection was performed using a ready‐to‐use streptavidin‐peroxidase (SP) kit (Zhongshan, Beijing, China), which includes biotinylated secondary antibodies and horseradish peroxidase‐conjugated streptavidin complex. Chromogenic development was carried out with 3,3'‐diaminobenzidine (DAB) substrate, followed by hematoxylin counterstaining and mounting for microscopic evaluation and image capture. Statistical analyses were performed using GraphPad Prism software (version 10; GraphPad Software, Boston, MA, USA). All experimental results were expressed as the mean ± standard deviation (SD). Differences between two independent groups were analyzed using the two‐tailed Student's t ‐test. For comparisons involving multiple experimental groups, one‐way analysis of variance (ANOVA) was conducted, followed by Tukey's post‐hoc tests for multiple comparisons correction. Statistical significance was defined as a p ‐value less than 0.05.