HAGLR promotes endometriosis progression via the miR-185-5p/VEGFA axis and serves as a potential diagnostic biomarker

110 patients diagnosed with endometriosis and treated at Shanghai Jiading District Anting Hospital between February 2019 and February 2022 were enrolled. All individuals were diagnosed via laparoscopy and confirmed by histopathological examination. These individuals constituted the endometriosis group. During the same period, 110 women undergoing physical examinations were recruited as the control group. Endometrial tissue samples were collected from all participants and immediately stored at − 80 °C for subsequent analysis. Disease staging was performed based on the revised classification system of the American Society for Reproductive Medicine (rASRM). Exclusion criteria: (1) Age < 18 years; (2) Presence of other gynecological disorders (e.g., ovarian malignancies); (3) Recent use of hormone therapy or other medications; (4) Postmenopausal status; (5) Cognitive impairment or diagnosed psychiatric disorders; (6) Presence of major organ dysfunction or malignancies. Written consent was secured from all participants. The Ethics Committee of Shanghai Jiading District Anting Hospital approved the study protocol (Approval No. 20190007). The human endometriotic epithelial 12Z cell line was obtained from Applied Biological Materials Co., Ltd (CA). Cells were cultured in DMEM/F-12 nutrient mixture (Gibco, USA) with the addition of 10% FBS (Gibco) and maintained at 37 °C and 5% CO 2 . Cells were passaged at 70–80% confluency using 0.25% trypsin-EDTA (Gibco). For functional experiments, 12Z cells received transfection of specific oligonucleotides or plasmids using Lipofectamine 3000 (Invitrogen, USA). Short hairpin RNA targeting HAGLR (sh-HAGLR), miR-185-5p inhibitor, and VEGFA overexpression plasmid (oe-VEGFA) were synthesized and constructed by GenePharma(Shanghai, China). Corresponding negative controls—sh-NC (non-targeting shRNA), inhibitor-NC (scrambled miRNA inhibitor), and oe-NC (empty vector)—were used as controls for each group. Briefly, 12Z cells were seeded into 6-well plates at a density of 2 × 10 5 cells/well and incubated overnight to reach 60–70% confluency. Transfection was performed with 100 nM oligonucleotides or 2 µg plasmid DNA per well using Lipofectamine 3000. After 6 h of incubation, the transfection medium was replaced with fresh complete medium, and cells were cultured for additional assays. The detailed sequences used for transfection are provided in Table S1 . Total RNA was extracted using TRIzol (Invitrogen). For the detection of HAGLR and VEGFA, 1 µg of total RNA was reverse-transcribed into cDNA using a PrimeScript RT reagent kit (Takara, Japan). For miR-185-5p detection, cDNA synthesis was performed using a miRNA-specific reverse transcription kit (Mir-X™ miRNA First-Strand Synthesis Kit; Takara). qRT-PCR was performed using SYBR Premix Ex Taq II (Takara). GAPDH and U6 were used as the reference genes. Relative quantification was performed using the 2 −ΔΔCt calculation. Protein extracts were prepared from cultured cells using RIPA lysis buffer supplemented with protease inhibitors (Beyotime, China). Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies against VEGFA (1:1000, Abcam, USA) and GAPDH (1:5000, Proteintech, China) at 4 °C overnight. Following incubation with HRP-conjugated secondary antibodies (1:4000, Proteintech), protein signals were detected using enhanced chemiluminescence. Band intensities were quantified using ImageJ software. The wild-type (WT) or mutant (MT) sequences of the predicted miR-185-5p binding sites in HAGLR and the 3′ untranslated region (3′UTR) of VEGFA were synthesized and cloned into the pmirGLO (Promega, USA), generating the reporter constructs WT-HAGLR, MT-HAGLR, WT-VEGFA, and MT-VEGFA. For the assay, 293 T cells were seeded into 24-well plates and co-transfected with 500 ng of luciferase reporter plasmid and 50 nM of miR-185-5p mimic or mimic negative control (mimic-NC) using Lipofectamine 3000. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, USA). Transfected 12Z cells were seeded into 96-well plates (3 × 10 3 cells/well). At each time point (at 0, 24, 48, and 72 h post-seeding), 10 µL of CCK-8 (Dojindo, Japan) was applied to every well, followed by incubation at 37 °C for 2 h. The absorbance was then measured at 450 nm via microplate reader (BioTek, USA). The proliferation curves were plotted based on the OD values at the indicated time points. Cell migration and invasion abilities were evaluated using Transwell chambers (Corning, USA). For the migration assay, 12Z cells were resuspended in serum-free medium, and 2 × 10 4 cells in 200 µL were seeded into the upper chamber. For the invasion, the upper surface of the membrane was pre-coated with 50 µL of Matrigel (BD Biosciences, USA) diluted 1:8 in serum-free medium, and 5 × 10 4 cells were seeded similarly. In both assays, 600 µL of medium containing 10% FBS was added to the lower chamber as a chemoattractant. After 24 h, non-migrated/non-invaded cells were removed, and the lower-side cells were fixed, stained with crystal violet, and imaged under a light microscope (Olympus, Japan). The number of stained cells was counted in five randomly selected fields, and the mean value was used for statistical analysis. All statistical analyses were performed using GraphPad Prism 9.0 and R 4.0.0. Data are expressed as mean ± standard deviation (SD). Differences between the two groups were assessed using Student’s t-test or chi-square test. For experiments involving more than two groups, one-way ANOVA was performed, followed by Bonferroni’s post hoc test to correct for multiple comparisons. Categorical variables were analyzed using the chi-square test. Receiver operating characteristic (ROC) curves, Pearson correlation analysis, and Logistic regression were used to assess diagnostic performance, linear correlations, and independent risk factors, respectively. P value < 0.05 is statistically significant.

The clinical profile of the EMs and control groups is summarized in Table  1 . No notable differences were observed in age, BMI, gravidity, parity, or menstrual cycle ( P  > 0.05). While the incidence of dysmenorrhea was higher in the EMs group ( P  < 0.05). Table 1 Baseline characteristics Parameters Control ( n  = 110) Endometriosis ( n  = 110) P -value Age, years 33.28 ± 7.76 32.54 ± 7.27 0.468 BMI, kg/m 2 23.63 ± 2.31 23.98 ± 2.37 0.263 Gravidity 1.68 ± 1.14 1.55 ± 1.11 0.405 Parity 1.12 ± 0.73 1.03 ± 0.70 0.348 Menstrual, days 28.55 ± 2.19 28.90 ± 2.70 0.299 Dysmenorrhea < 0.001  Present 21 68  Absent 89 42 rASRM endometriosis stage  Ⅰ/Ⅱ - 47  Ⅲ/Ⅳ - 63 DIE  No - 68  Yes - 42 Abbreviations : BMI Body mass index, DIE Deeply infiltrating endometriosis, rASRM  revised American Society of Reproductive Medicine Baseline characteristics Abbreviations : BMI Body mass index, DIE Deeply infiltrating endometriosis, rASRM  revised American Society of Reproductive Medicine HAGLR expression was significantly elevated in EMs tissues, with approximately a 40% increase (Fig.  1 A, P  < 0.05). ROC curve demonstrated its diagnostic potential, yielding an AUC of 0.83, with a sensitivity of 0.90 and specificity of 0.70 (Fig.  1 B). Logistic regression analysis identified HAGLR, along with dysmenorrhea, as an independent risk factor for EMs (Fig.  1 C, P  < 0.05). Based on the median HAGLR expression level, EMs participants were grouped into high and low levels groups. Higher HAGLR was significantly associated with DIE (Table  2 , P  = 0.0308) and advanced rASRM stage (Table  2 , P  = 0.0207). Although dysmenorrhea was more frequent in the high HAGLR group, the observed difference lacked statistical significance ( P  = 0.077). Fig. 1 Aberrant expression of HAGLR in 220 clinical samples ( n  = 110 for the EMs group; n  = 110 for the control group). A  HAGLR expression was elevated in the EMs group ( n  = 110) versus control tissues ( n  = 110). B  ROC curve analyzed the diagnostic potential of miR-191-5p. C  Logistic regression analysis indicated that HAGLR was an independent risk factor. *** P  < 0.001 vs. EMs group. EMs, Endometriosis Aberrant expression of HAGLR in 220 clinical samples ( n  = 110 for the EMs group; n  = 110 for the control group). A  HAGLR expression was elevated in the EMs group ( n  = 110) versus control tissues ( n  = 110). B  ROC curve analyzed the diagnostic potential of miR-191-5p. C  Logistic regression analysis indicated that HAGLR was an independent risk factor. *** P  < 0.001 vs. EMs group. EMs, Endometriosis Table 2 Relationship between the expression of HAGLR and clinicopathological features in endometriosis Features Cases ( n  = 110) Low HAGLR ( n  = 55) High HAGLR ( n  = 55) P value Dysmenorrhea 0.077  Absent 42 26 16  Present 68 29 39 rASRM endometriosis stage 0.0207  Ⅰ/Ⅱ 47 30 17  Ⅲ/Ⅳ 63 25 38 DIE 0.0308  No 68 40 28  Yes 42 15 27 Abbreviations : DIE Deeply infiltrating endometriosis, rASRM  revised American Society of Reproductive Medicine Relationship between the expression of HAGLR and clinicopathological features in endometriosis Abbreviations : DIE Deeply infiltrating endometriosis, rASRM  revised American Society of Reproductive Medicine To explore the functional role of HAGLR in EMs, 12Z cells were transfected with shRNA targeting HAGLR. qRT-PCR confirmed effective knockdown, with over 50% reduction in expression (Fig.  2 A, P  < 0.05). Functional assays showed that HAGLR knockdown significantly reduced cell migration (Fig.  2 B, P  < 0.05), invasion (Fig.  2 C, P  < 0.05), and proliferation (Fig.  2 D, P  < 0.05). Fig. 2 HAGLR promoted EC cell proliferation, migration, and invasion. A  HAGLR was reduced following sh-HAGLR transfection. B-D  Knockdown of HAGLR inhibited 12Z cell migration ( B ), invasion ( C ), and proliferation ( D ). * P  < 0.05, ** P  < 0.01, *** P  < 0.001 vs. Control HAGLR promoted EC cell proliferation, migration, and invasion. A  HAGLR was reduced following sh-HAGLR transfection. B-D  Knockdown of HAGLR inhibited 12Z cell migration ( B ), invasion ( C ), and proliferation ( D ). * P  < 0.05, ** P  < 0.01, *** P  < 0.001 vs. Control Bioinformatic analysis using StarBase predicted a direct binding relationship between HAGLR and miR-185-5p, with the binding sites illustrated in Fig.  3 A. miR-185-5p mimic decreased luciferase activity in the WT-HAGLR group, but not in the MT group, confirming direct targeting (Fig.  3 B, P  < 0.05). miR-185-5p abundance was downregulated in EMs tissues (Fig.  3 C, P  < 0.05) and negatively correlated with HAGLR levels (Fig.  3 D, P  < 0.05). Transfection with a miR-185-5p inhibitor effectively suppressed its expression ( P   0.05). Functional rescue experiments demonstrated that miR-185-5p inhibition restored the proliferative, migratory, and invasive capabilities of 12Z cells following HAGLR knockdown (Fig.  3 F-G, P  < 0.05). This evidence implies that HAGLR promotes EMs cell behaviors by suppressing miR-185-5p. Fig. 3 HAGLR targeted the miR-185-5p. A  Predicted binding sites between HAGLR and miR-185-5p. B  Dual-luciferase reporter assay confirmed the interaction between HAGLR and miR-185-5p in WT but not MUT constructs ( n  = 3). C  miR-185-5p was reduced in the EMs tissues ( n  = 110) compared to control tissues ( n  = 110). D  A negative correlation between HAGLR and miR-185-5p expression ( n  = 110). E  miR-185-5p levels were reduced after co-transfection with shRNA and miR-inhibitor in 12Z cell lines ( n  = 3). F-G  Inhibitor of miR-185-5p promoted 12Z cell migration/invasion ( F ) and proliferation ( G ). * P  < 0.05, ** P  < 0.01, *** P  < 0.001 vs. EMs, Endometriosis HAGLR targeted the miR-185-5p. A  Predicted binding sites between HAGLR and miR-185-5p. B  Dual-luciferase reporter assay confirmed the interaction between HAGLR and miR-185-5p in WT but not MUT constructs ( n  = 3). C  miR-185-5p was reduced in the EMs tissues ( n  = 110) compared to control tissues ( n  = 110). D  A negative correlation between HAGLR and miR-185-5p expression ( n  = 110). E  miR-185-5p levels were reduced after co-transfection with shRNA and miR-inhibitor in 12Z cell lines ( n  = 3). F-G  Inhibitor of miR-185-5p promoted 12Z cell migration/invasion ( F ) and proliferation ( G ). * P  < 0.05, ** P  < 0.01, *** P  < 0.001 vs. EMs, Endometriosis StarBase analyses predicted miR-185-5p binding sites within VEGFA (Fig.  4 A). Dual-luciferase assay confirmed this interaction, as miR-185-5p mimic reduced luciferase activity in WT-VEGFA but not in MT-VEGFA constructs (Fig.  4 B, P  < 0.05). VEGFA was elevated in EMs group (Fig.  4 C, P  < 0.05). Moreover, an inverse correlation was observed between miR-185-5p and VEGFA, whereas HAGLR and VEGFA were positively correlated (Fig.  4 D-E, P  < 0.05). Co-transfection with sh-HAGLR and oe-VEGFA significantly increased VEGFA expression (Fig.  4 F, P   0.05). VEGFA protein levels by Western blot. The results showed that VEGFA protein expression followed a pattern consistent with the mRNA data, supporting the reliability of our findings (Fig.  4 G, P  > 0.05). Rescue assays showed that overexpression of VEGFA enhances the effects on cell migration, invasion (Fig.  4 H, P  < 0.05), and proliferation (Fig.  4 I, P  < 0.05). Together, these findings support the notion that lncRNA HAGLR contributes to the advancement of EMS by modulating the miR-185-5p/VEGFA, thereby enhancing cell growth and metastatic potential. Fig. 4 VEGFA was a direct target of miR-185-5p. A  Predicted binding sites between VEGFA and miR-185-5p. B  Dual-luciferase reporter assay confirmed the interaction between VEGFA and miR-185-5p in WT but not MUT constructs ( n  = 3). C  VEGFA was elevated in the EMs tissues ( n  = 110) compared to control tissues ( n  = 110). D  A negative correlation between HAGLR and miR-185-5p expression ( n  = 110). E  VEGFA was positively linked to miR-185-5p levels. F  VEGFA levels were increased after co-transfection with shRNA and oe-VEGFA in 12Z cell lines ( n  = 3). G  The relative protein level of VEGFA. H-I  Overexpression of VEGFA activated 12Z cell migration/invasion ( H ) and cell proliferation ( I ). * P  < 0.05, ** P  < 0.01, *** P  < 0.001 vs. EMs, Endometriosis VEGFA was a direct target of miR-185-5p. A  Predicted binding sites between VEGFA and miR-185-5p. B  Dual-luciferase reporter assay confirmed the interaction between VEGFA and miR-185-5p in WT but not MUT constructs ( n  = 3). C  VEGFA was elevated in the EMs tissues ( n  = 110) compared to control tissues ( n  = 110). D  A negative correlation between HAGLR and miR-185-5p expression ( n  = 110). E  VEGFA was positively linked to miR-185-5p levels. F  VEGFA levels were increased after co-transfection with shRNA and oe-VEGFA in 12Z cell lines ( n  = 3). G  The relative protein level of VEGFA. H-I  Overexpression of VEGFA activated 12Z cell migration/invasion ( H ) and cell proliferation ( I ). * P  < 0.05, ** P  < 0.01, *** P  < 0.001 vs. EMs, Endometriosis

Endometriosis (EMs) is a prevalent gynecological disorder marked by the occurrence of endometrial glands and stroma at extrauterine sites [ 1 – 4 ]. Despite its benign nature, ectopic endometrial cells exhibit malignant-like behaviors, including uncontrolled proliferation, local invasion, and distant dissemination [ 5 , 6 ]. EMs primarily affect reproductive-aged women, with epidemiological studies indicating a prevalence of over 10% [ 6 ]. The disease manifests in heterogeneous forms and is commonly classified into: superficial peritoneal lesions, ovarian endometriomas, and deep infiltrating endometriosis (DIE) [ 7 ]. As EMs progress, patients often experience clinical symptoms such as secondary dysmenorrhea and dyspareunia [ 8 ]. However, due to the nonspecific and often subtle early symptoms, timely diagnosis remains a significant challenge [ 9 ]. The precise etiology and underlying pathophysiological mechanisms of EMs remain elusive, and no curative treatments are currently available. Therefore, elucidating the molecular mechanisms involved in EMs pathogenesis and identifying novel therapeutic targets are critical research priorities. Long non-coding RNAs (lncRNAs), transcripts longer than 200 nucleotides lacking protein-coding capacity, have been recognized as crucial regulators of gene expression at transcriptional, post-transcriptional, and epigenetic levels [ 10 ]. lncRNAs can act as competing endogenous RNAs (ceRNA), sponging microRNAs (miRNAs) and thereby modulating the expression of miRNA target genes [ 11 ]. The regulatory interactions within lncRNA/miRNA/mRNA networks are gaining attention in EMs research. For example, lncRNA BANCR promotes stromal cell motility and invasiveness by regulating the miR-15a-5p/TRIM59 axis, aggravating EMs progression [ 12 ]. Similarly, HOTAIR has been identified as a pathogenic lncRNA in EMs; its downregulation suppresses stromal cell growth and invasion via the miR-519b-3p/PRRG4 axis [ 13 ]. HAGLR, a lncRNA located at chromosome 2q31.1, has emerged as a possible biomarker in various pathological conditions [ 14 – 16 ]. In EMs, HAGLR was identified as a candidate regulatory lncRNA through ceRNA network analysis of publicly available sequencing datasets, with GEO data revealing significant upregulation of HAGLR expression in EMs tissues [ 17 , 18 ]. The abnormal expression of HAGLR in ectopic endometrium was further validated using the GSE105764 dataset (Figure S1). Nevertheless, the specific biological roles and governing mechanisms of HAGLR in EMs remain largely unexplored. Notably, our previous bioinformatic analysis predicted direct binding sites between HAGLR and miR-185-5p, a miRNA found to be significantly reduced in EMs [ 19 ]. Based on this, we hypothesize that HAGLR may promote EMs progression through modulation of the miR-185-5p, a hypothesis warranting further experimental validation. In summary, this study investigates the expression and functional role of the HAGLR/miR-185-5p regulatory axis in EMs using cell-based models. The findings offer novel insights into the molecular mechanisms underlying EMs and may contribute to the development of targeted therapeutic interventions.

EMs remains a clinical challenge. Genomic studies indicate that many EMs-associated risk loci are located in non-coding regions, underscoring the potential involvement of lncRNAs in disease pathogenesis [ 20 ]. In this study, HAGLR was identified as an independent risk factor for EMs and was upregulated in EMs tissues, aligning with previous findings from GEO datasets [ 18 ]. Dysmenorrhea, a common symptom of EMs [ 21 ], was reported by nearly two-thirds of our cohort. While high HAGLR expression appeared more frequent in patients with dysmenorrhea, the association did not reach statistical significance, potentially due to sample size limitations. Clinically, rASRM staging system is widely used to assess EMs severity, grading lesions based on extent, depth, and adhesions [ 22 , 23 ]. Among EMs subtypes, DIE is the most aggressive, characterized by lesion invasion into pelvic organs, often resulting in progressive functional impairment [ 7 , 24 ]. Our findings revealed that elevated HAGLR expression correlated with both the DIE subtype and higher rASRM stages. Taken together with ROC analysis and clinical associations, HAGLR may serve as a promising diagnostic biomarker and play a key role in the progression of EMs. Endometriosis is a complex condition with tumor-like biological behavior [ 6 ]. The 12Z endometrial stromal cell line, an immortalized model, is commonly used to investigate the molecular mechanisms of EMs and to validate potential pathogenic genes [ 25 , 26 ]. Since endometrial stromal cells (ESCs) are central to EMs pathogenesis—driving lesion formation, invasion, and pain [ 27 ]—HAGLR is silenced in 12Z cells to assess its functional role in cellular behavior. The viability and motility of ESCs are critical to EMs progression. Enhanced proliferative capacity allows ectopic endometrial tissue to expand in abnormal locations, while migratory and invasive properties facilitate lesion establishment and spread [ 7 , 28 ]. As lesions enlarge, they can compress or adhere to surrounding organs, exacerbating clinical symptoms [ 29 ]. Herein, HAGLR knockdown reduced the proliferation and invasion of 12Z cells, indicating that HAGLR contributes to the aggressive cellular phenotype observed in EMs. Based on findings, HAGLR plays a pathogenic role in disease development and may represent a potential candidate for therapeutic intervention. Recent studies have increasingly emphasized the importance of the lncRNA/miRNA/mRNA regulatory network in EMs. lncRNAs can function as molecular “sponges,” competitively binding to miRNAs and attenuating their inhibitory effects on downstream target mRNAs [ 11 ]. However, its specific regulatory mechanisms in EMs remain largely unexplored. Similar to the findings of this study, HAGLR-an upregulated lncRNA in EMs-may exert its function by competitively binding to miRNAs. Bioinformatic predictions and experimental validation confirmed that miR-185-5p is a direct target of HAGLR. Consistent with previous reports [ 19 ], in our cohort, miR-185-5p is reduced in EMs group. Moreover, miR-185-5p expression was shown to decrease in 12Z cell lines following treatment with peritoneal fluid from EMs patients [ 30 ], further supporting its dysregulation in the disease. Functional assays revealed that miR-185-5p downregulation enhanced 12Z cell proliferation and invasion, indicating its suppressive role in EMs. Subsequent analysis identified VEGFA as a downstream target of miR-185-5p, which was also validated experimentally. VEGFA has previously been shown to be upregulated in 12Z cells [ 31 ], and its expression increases with disease severity in DIE [ 32 ], aligning with our findings. Upregulation of VEGFA significantly promoted 12Z cell growth, motility, and invasiveness. Collectively, HAGLR facilitates EMs progression by sponging miR-185-5p and upregulating VEGFA, thereby promoting ESC aggressiveness. As a key regulator of angiogenesis, VEGFA supports lesion survival and expansion by enhancing vascularization [ 33 ]. It is reported that VEGFA inhibition using small molecules has been shown to reduce the growth of endometriotic implants in murine models [ 34 ]. Given its critical role, anti-angiogenic therapies targeting VEGFA represent a promising direction for EMs treatment. Although the biological functions of lncRNA HAGLR have been investigated in several diseases, only one previous study has linked HAGLR to endometriosis, and its mechanistic role in this condition remains largely unexplored. In particular, the interactions between HAGLR and specific miRNAs, as well as its potential contribution to the pathological behaviors of endometriotic epithelial cells, have not been reported. Given the complexity and heterogeneity of endometriosis, clarifying the regulatory axis involving HAGLR is essential for improving our understanding of disease progression and identifying potential molecular targets for diagnosis and therapy. Nevertheless, this study has several limitations. Although qRT-PCR was used to quantify the expression levels of HAGLR, miR-185-5p, and VEGFA, spatial localization validation would further strengthen the conclusions. We attempted RNA in situ hybridization (ISH) to detect HAGLR; however, the signals were extremely weak or undetectable, likely due to low endogenous abundance and technical challenges related to probe accessibility. Similar difficulties were encountered when attempting in situ hybridization for miR-185-5p. Due to current platform limitations and cost constraints, optimized ISH experiments could not be included in this study, and the results remain inconclusive. Additional in vivo evidence and tissue-level localization data would be valuable to further support the proposed regulatory mechanism. These aspects will be prioritized in our future work, along with improving probe design and hybridization conditions to enable reliable detection of low-abundance RNAs. Additionally, this study primarily employed a knockdown model to investigate the functional role of HAGLR. Although this approach is clinically relevant given the marked upregulation of HAGLR in endometriotic lesions, we were unable to achieve adequate overexpression efficiency in preliminary tests. As a result, overexpression experiments were not included. Future studies will incorporate optimized overexpression systems to further validate the bidirectional regulatory role of HAGLR and strengthen the mechanistic conclusions. While our study identifies HAGLR/miR-185-5p/VEGFA as a key regulatory axis in endometriosis, the broader ceRNA network regulated by HAGLR remains incompletely characterized. Future work integrating multi-omics approaches-such as lncRNA/miRNA/mRNA co-expression networks and AGO2-CLIP-seq-will be important for identifying additional interacting molecules and potential compensatory pathways. Moreover, we cannot exclude the possibility that HAGLR exerts functions beyond ceRNA activity, including modulation of signaling pathways or direct protein interactions. Techniques such as CHIRP-MS, RNA pulldown, and CRISPR-based genomic perturbation will be valuable for delineating the full functional landscape of HAGLR in endometriosis. Another limitation of this study is that the functional experiments were conducted exclusively in the immortalized human endometriotic epithelial 12Z cell line. Although 12Z cells provide a stable and well-established model for mechanistic investigations, they may not fully recapitulate the biological characteristics of primary endometrial or endometriotic cells. The limited availability, variable viability, and lower transfection efficiency of primary cells prevented us from performing parallel validation experiments in this study. Future research will attempt to incorporate primary endometrial stromal and epithelial cells to further validate the phenotypic effects of the HAGLR/miR-158-5p/VEGFA axis and enhance the clinical relevance of our findings. One limitation of this study is the absence of in vivo validation to assess the effects of miR-191-5p. Although preliminary plans for animal experiments were considered, they could not be implemented due to constraints in funding and technical resources. Future research will aim to include animal models to elucidate the in vivo mechanisms of HAGLR in EMs progression.

In conclusion, this study demonstrates that HAGLR is significantly overexpressed in endometriosis and serves as a promising diagnostic biomarker. HAGLR promotes the proliferation and metastatic behaviors of ESCs by modulating the miR-185-5p/VEGFA, thereby contributing to disease progression. These findings highlight HAGLR as a potential therapeutic target, providing a theoretical foundation for future clinical research and drug development in endometriosis.

Supplementary Material 1 Supplementary Material 1 Supplementary Material 2 Supplementary Material 2