Endometriosis-derived exosomal MicroRNA-125b-5p downregulates phosphatidylinositol 3-Kinase/Protein Kinase B signaling pathways via vascular endothelial growth factor to decelerate endometriosis progression

A total of 25 EMs patients and 24 non-EMs patients were recruited from the Department of Gynecology of Shenzhen Maternity and Child Healthcare Hospital from July 2019 to April 2021. All participants were fully informed of the study procedures and provided written informed consent. The protocol was approved by the Ethics Committee of Shenzhen Maternity and Child Healthcare Hospital (No:20191239). Clinic data were collected, including age, body mass index (BMI), visual analogue scale (VAS) dysmenorrhea score, maximum cyst diameter, number of pregnancies, and parity (Supplement Table 1). General Inclusion Criteria (Applied to both groups): i: participants were aged between 20 and 45 years; ii: no hormone therapy for at least 3 months prior to sample collection; iii: absence of other major systemic diseases. Specific Criteria for the EMs Group: confirmation of endometriosis by laparoscopy and subsequent histopathology examination, with disease stage > 3. Specific Criteria for the non-EMs Control Group: patients underwent laparoscopic surgery for tubal obstructive infertility with no visual evidence of endometriosis. These patients were also confirmed to be free of other non-inflammatory benign diseases (e.g., adenomyosis, uterine fibroids, polyps). The sample from 4 EMs patients and 3 non-EMs patients were used for exosome small RNA sequencing, while the remaining samples were used for Q-PCR detection. Because cyst fluid contains a large number of impurities, ultra-high-speed centrifugation alone cannot isolate high quality exosomes from cyst fluid. Therefore, an optimized protocol was used to isolate high-quality exosomes from pelvic fluid(PF) of non-EMs patients (Ctr1, Ctr2, and Ctr3) or cyst fluid(CF) of EMs patients (PF1, PF2, PF3 and PF4 from EMs patients’ pelvic fluid; CF1, CF2, CF3 and CF4 from EMs patients’ cyst fluid). Briefly, 6 mL PF or CF were collected and centrifugated at 500×g for 10 min. The supernatant was collected and centrifugated at 2000×g for 10 min, followed by centrifugation at 10,000×g for 20 min twice. Then, the resulting supernatant was collected and mix with an equal volume of 4% polyethlene glycol 4000 (Sangon Biotech, Shanghai, China) for 5 min and centrifugated at 10,000×g for 5 min. Next, the supernatant was then subject to sequential centrifugation at 13,000×g for 30 min, and at 100,000×g for 70 min (Hitachi, Tokyo, Japan) continued. The pellets were suspended in 300 µL of phosphate-buffered saline (PBS), and centrifuged again at 100,000×g for 70 min, collected pellets and suspended by 100 µL PBS. All procedures were performed at 4 °C.Transmission electron microscopy (TEM) images were obtained using a Hitachi Electron Microscope H-600. The sizes of exosomes were measured using NanoSight 300 analysis (Malvern Panalytical, Malvern, UK), while exosome specific protein markers (CD63 and TSG101) were detected by western blot. Exosomal RNA was isolated using the QIAGEN exoRNeasy Serum/Serum Maxi Kit (QIAGEN, Hilden, Germany). RNA quality and quantity were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). Small RNA sequencing library was constructed with a QIAseq miRNA Library Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Small RNA sequencing was performed by using the SE75 mode the Illumina NextSeq 500 sequencing platform (Illumina, San Diego, USA). After data cleaning, sequences > 18 nucleotides (nt) were aligned against miRBase (v22). miRNA expression levels were normalized using reads per million mappable miRNA sequences. Differential expression analysis between EMs and non-EMs groups was performed using the DESeq2 R-package. miRNAs with fold change (FC) ≥ 2 ( p  < 0.05) were considered differentially expressed. Target gene prediction was performed with online bioinformatics tools (miRanda and RNA hybrid). The obtained target genes were analyzed by GO function analysis ( http://www.geneontology.org/ ) and KEGG pathway analysis ( www.kegg.jp/kegg/kegg1.html ). A GO term with corrected p ⩽0.05 and a pathway with Q ⩽0.05 were considered significant enriched. Q-PCR was used to validate eight significantly differentially expressed miRNAs identified by small RNA sequencing. Primer sequences are provided in Supplement Table 2. All reactions were performed using a SYBR Green Master Mix (TaKaRa, Shiga, Japan). PCR amplification was conducted in a total volume of 20 µL as follows: 95 °C for 5 min, followed by 40 cycles of 95 °C for 5 s, 55 °C for 30 s, and 72 °C for 30 s. All reactions were performed in triplicate. The miRNA expression levels were normalized to U6, and relative transcription levels were calculated using the comparative threshold cycle values method (2-ΔΔCt). Each measurement was performed in three biological replicates. Primary ectopic human endometrial stromal cells (HESCs) were isolated from endometriosis lesions, and cultured in DMEM/F-12 medium supplemented with 5% FBS, 50 µg/mL penicillin, and 50 µg/mL streptomycin. To induce in vitro differentiation, the cells were treated with a DC containing 0.5 mM 8-Br-cAMP. When cells reached about 70% confluency, they were seeded into a 6-well plate at a density of 3*10 4 / well. miR-125b-5p NC, miR-125b-5p-mimics and miR-125b-5p-inhibitor (GenePharma, Suzhou, China, sequence are shown in Supplement Table 3) were transfected into HESCs using Lipofectamine™ 3000 transfection reagent (Invitrogen, L3000001). Q-PCR was used to detect PI3K and Akt mRNA experssion (Primers are shown in Supplement Table 2). Western blot was used to detecte and the protein expression of PI3K (PI3K antibody, GTX100462, GeneTex) and Akt (Akt antibody, GTX128414, GeneTex). After 48 h of transfection, cell Counting Kit-8 (C0038, Beyotime, China) was used to assess HESCs proliferation. Migration assay was done in 8 μm pore Transwell chambers (Corning, NY, United States), and invasion assays were performed in Transwell chambers with Matrigel (BD Biosciences, MA, United States). The online starBase software ( http://starbase.sysu.edu.cn/ ) was used to predict the binding sites between miR-125b-5p and the 3′ untranslated regions of VEGF mRNA. miR-125b-5p mimic, miR-125b-5p-inhibitor and negative controls were synthesized by GenePharma Co., Ltd.(Suzhou, China, sequence are shown in Supplement Table 3), and cloned into a pGL3 vector and pLenti-CMV-GFP vector. HUVEC cells were cultured at 37℃ with 5% CO2. Cells were transfected using Lipofectamine 2000. After incubation for 48 h, total RNAs and total proteins were extracted. VEGFmRNA and protein levels were detected by qPCR and Western blot, respectively. Luciferase activity was measured using a luciferase detection kit (Beyotime, Shanghai, China) Each measurement was performed in three biological replicates. Statistical analyses were performed using SPSS 26.0 (SPSS Inc., Chicago, IL, USA). Data are expressed as mean±standard deviation. Clinical information and miRNA expression profiles were analyzed using independent sample t-tests, and differences were considered significant at p  < 0.05. Receiver Operating Characteristic (ROC) analysis and areas under the curve (AUC) were calculated to assess the diagnostic power of EMs. AUC = 0.5 indicates no predictive power, whereas AUC = 1 represents perfect predictive performance.

The morphological features of exosomes were observed by TEM. As shown in Fig.  1 A, the particles were spherical structures and most of the round microvesicles were 90 ~ 120 nm in diameter. The sizes of isolate particles ranged from 39 nm to 170 nm in diameter and concentrations of isolate particles were 1.73*10 10 particles/ml, 3.21*10 10 particles/ml, respectively (Fig.  1 B). In addition, exosome specific protein markers (CD63, TSG101) were detected by western blot (Fig.  1 C). These results indicated that our protocol is able to isolated exosomes successfully from cyst fluid and pelvic fluid. Fig. 1 Characterization of cyst fluid and pelvic fluid exosomes; ( A ) TEM images of exosomes (scale bar: 500 nm); ( B ) Size distribution and concentrations of exosomes; ( C ) The expression of the exosomes specific markers of TSG101and CD63 Characterization of cyst fluid and pelvic fluid exosomes; ( A ) TEM images of exosomes (scale bar: 500 nm); ( B ) Size distribution and concentrations of exosomes; ( C ) The expression of the exosomes specific markers of TSG101and CD63 Eleven small RNA sequencing datasets were generated via high-throughput sequencing. A total of 158 miRNAs differentially expressed in exosomes when comparing the control group with the CF group, including 118 upregulated and 40 downregulated miRNAs. Using the same criteria, 22 upregulated and 32 downregulated miRNAs were identified when comparing the control group with the PF group. In addition,133 upregulated and 55 downregulated miRNAs were identified when comparing the PF group with the CF group (Fig.  2 A and B). Hierarchical cluster analysis showed the the expression patterns of top 40 most highly expressed known miRNAs in each sample (Fig.  2 C). The four most downregulated miRNAs in EMs cyst fluid were miR-125b-5p, miR-328-3p, miR-125a-5p, miR-30e-3p, whereas the four most upregulated miRNAs were miR-3141,miR-223-3p, miR-142-5p, miR-1246 (Supplement Table 4). Fig. 2 Analysis of differentially expressed miRNAs. ( A ) Volcano plot analysis of differentially expressed miRNAs between the control and CF group, control and PF group, PF and CF group. The green points represent down-regulated miRNA; the grey points represent no significant miRNA; the red points represent up-regulated miRNA. ( B ) Differentially expressed miRNA statistical between the control and CF group, control and PF group, PF and CF group. ( C ) Heatmap of hierarchical clustering analysis of top 40 highest expressions of known miRNAs in each sample. Green represents low expression; red represents high expression Analysis of differentially expressed miRNAs. ( A ) Volcano plot analysis of differentially expressed miRNAs between the control and CF group, control and PF group, PF and CF group. The green points represent down-regulated miRNA; the grey points represent no significant miRNA; the red points represent up-regulated miRNA. ( B ) Differentially expressed miRNA statistical between the control and CF group, control and PF group, PF and CF group. ( C ) Heatmap of hierarchical clustering analysis of top 40 highest expressions of known miRNAs in each sample. Green represents low expression; red represents high expression GO and KEGG annotation and enrichment analyses were performed to identify the functions and mechanisms of differentially expressed miRNAs target genes. The function and pathway of differentially expressed miRNAs between the control and CF group were mainly associated with cellular process, biological regulation and metabolic process in GO annotation analysis (Fig.  3 A). KEGG pathway analysis indicated that these miRNAs were primarily enriched in viral infectious disease pathways and cancer-related pathways (Fig.  3 B). Further GO and KEGG enrichment analysis demonstrated that the differentially expressed miRNAs were mainly involved in neurological pathway and blood vessel formation (Fig.  3 C and D). Fig. 3 Function and pathway analysis of the target genes of differentially expressed miRNAs in exosomes. ( A , C ) GO enrichment analysis of the target genes of differentially expressed miRNAs between the control and CF group. ( B , D ) KEGG enrichment analysis of the target genes of differentially expressed miRNAs between the control and CF group ( www.kegg.jp/kegg/kegg1.html ). The horizontal axis represents the enrichment rate, the size of the dot represents the number of genes, and the color of the dot represents p value Function and pathway analysis of the target genes of differentially expressed miRNAs in exosomes. ( A , C ) GO enrichment analysis of the target genes of differentially expressed miRNAs between the control and CF group. ( B , D ) KEGG enrichment analysis of the target genes of differentially expressed miRNAs between the control and CF group ( www.kegg.jp/kegg/kegg1.html ). The horizontal axis represents the enrichment rate, the size of the dot represents the number of genes, and the color of the dot represents p value The expression levels of the eight candidate miRNAs in each sample were showed in Fig.  4 A. First, we screened the eight candidature miRNA in serum sample from 7 volunteers (4 EMs patients and 3 patients who were to use for miRNA sequencing) by Q-PCR (Fig.  4 B), the levels of eight candidate miRNA were similar to the sequencing results. Comparing to the control group, the expression levels of miR-125b-5p, miR-328-3p, miR-125a-5p, miR-30e-3p were decreased, whereas miR-3141, miR-223-3p, miR-142-5p, miR-1246 were increased, but miR-328-3p, miR-3141 and miR-142-5p had not significant difference ( p  > 0.05). To further validate the clinical potential of these candidates as non-invasive biomarkers, we quantified their expression in serum samples from an independent cohort comprising 21 EMs patients and 21 matched non-EMs controls (Fig.  4 C). Comparing to non-EMs group, the expression levels of miR-125a-5p, miR-30e-3p were decreased in EMs group, whereas miR-3141, miR-223-3p, miR-142-5p, miR-1246 were increased ( p  < 0.05). Meanwhile, the expression of miR-125b-5p in serum was remarkably downregulated in the EMs group ( p  < 0.01). These observations suggest that these miRNAs are involved in EMs and may has a strong correlation with EMs. Moreover, ROC found that miR-125b-5p was the most valuable miRNA for distinguishing EMs (AUC = 0.925; 95% CI, 0.85 ~ 1) (Fig.  4 D). Although miR-125b-5p was significantly downregulated in cyst fluid and serum, it is worth noting that we observed no statistically significant difference in miR-125b-5p expression in the PF between EMs and non-EMs patients (Supplementary Table 5). This suggests that miR-125b-5p was high relationship with EMs endometriosis pathogenesis. Fig. 4 The expression of candidate miRNAs in serum by RT-PCR. A : candidate miRNAs expression in high-throughput sequencing sample; B : The expression levels of eight candidate miRNAs in each sample. C : candidate miRNAs expression in validate sample ( n  = 21); D: ROC analysis results. * p  < 0.05, ** p  < 0.01 The expression of candidate miRNAs in serum by RT-PCR. A : candidate miRNAs expression in high-throughput sequencing sample; B : The expression levels of eight candidate miRNAs in each sample. C : candidate miRNAs expression in validate sample ( n  = 21); D: ROC analysis results. * p  < 0.05, ** p  < 0.01 The function of miR-125b-5p was further evaluated by silencing or overexpressing miR-125b-5p in HESCs. After transfection with miR-125b-5p-mimics or miR-125b-5p-inhibitor, miR-125b-5p levels were increased or decreased in HESCs cells (Fig.  5 A). Silence of miR-125b-5p enhanced cell proliferation while overexpression of miR-125b-5p suppressed cell proliferation (Fig.  5 B) when compared with the controls. Moreover, as shown in Fig.  5 C, miR-125b-5p-mimics reduced the migration of HESCs cells in Transwell assays, by contrast, miR-125b-5p-inhibitor had more migration of HESCs cells. In addition, less HESCs cells invaded through the Matrigel in miR-125b-5p-mimics and more in miR-125b-5p-inhibitor in Boyden assays (Fig.  5 D), which suggesting overexpression of miR-125b-5p suppressed cell migration and invasion while silence of miR-125b-5p enhanced cell migration and invasion. Fig. 5 The biological function of HESCs cell after treat with miR-125b-5p mimic and inhibitor. ( A ) mRNA levels of miR-125b-5p in HUVSCs measured by qPCR ( B ). Results of CCK8 assays of proliferation ( C ) Representative images of migration by Transwell assay after 48 h of transfection ( D ) Representative images of invasion by Boyden assay after 48 h of transfection (* p  < 0.05, ** p  < 0.01,*** p  < 0.01) The biological function of HESCs cell after treat with miR-125b-5p mimic and inhibitor. ( A ) mRNA levels of miR-125b-5p in HUVSCs measured by qPCR ( B ). Results of CCK8 assays of proliferation ( C ) Representative images of migration by Transwell assay after 48 h of transfection ( D ) Representative images of invasion by Boyden assay after 48 h of transfection (* p  < 0.05, ** p  < 0.01,*** p  < 0.01) GO and KEGG enrichment analysis showed differentially expressed miRNAs may involve blood vessel formation. VEGF was the most important promoter factor in angiogenesis. To investigate if exosomal miR-125b-5p correlated with angiogenesis, we used HUVSCs as an in vitro model. The Matrigel-based tube formation assays indicated that overexpression of miR-125b-5p inhibited angiogenesis, while silencing of miR-125b-5p promoted angiogenesis (Fig.  6 A). Meanwhile, qPCR analysis showed VEGF mRNA levels were significantly decreased in miR-125b-5p-mimic group, but its expression was increased in miR-125b-5p -inhibitor group (Fig.  6 B). Western blot analysis showed miR-125b-5p mimic transfection can decrease protein levels of VEGF (Fig.  6 C). We predicted the binding sites of miR-125b-5p and VEGF as shown in the Fig.  6 D. Dual luciferase reporter assay results showed miR-125b-5p mimic significantly decreased the luciferase activity for VEGF mRNA (Fig.  6 E). These results established a direct regulatory relationship between miR-125b-5p on VEGF and demonstrated increased miR-125b-5p was down-regulation VEGF. Fig. 6 The levels of VEGF after treat with miR-125b-5p in HUVSCs cell. ( A ) Matrigel-based tube formation assays results in HUVSCs ( B ) mRNA levels of VEGF in HUVSCs measured by qPCR ( C ) Protein levels of VEGF in HUVSCs measured by western blot and image analysis ( D ) Binding sites of miR-125b-5p and VEGF ( E ) Dual luciferase reporter gene system assay was performed to validate the binding sites of miR-125b-5p and VEGF * p  < 0.05, ** p  < 0.01,*** p  < 0.01) The levels of VEGF after treat with miR-125b-5p in HUVSCs cell. ( A ) Matrigel-based tube formation assays results in HUVSCs ( B ) mRNA levels of VEGF in HUVSCs measured by qPCR ( C ) Protein levels of VEGF in HUVSCs measured by western blot and image analysis ( D ) Binding sites of miR-125b-5p and VEGF ( E ) Dual luciferase reporter gene system assay was performed to validate the binding sites of miR-125b-5p and VEGF * p  < 0.05, ** p  < 0.01,*** p  < 0.01) Q-PCR results showed that the expression of PI3K and AKT mRNA was downregulated in miR-125b-5p-overexpressing HESCs cells (Fig.  7 A). Meanwhile, PI3K and AKT mRNA were upregulated in miR-125b-5p-silencing HESCs cells (Fig.  7 A). Moreover, the PI3K and AKT proteins levels were also consistent with qPCR (Fig.  7 B and C), indicating that miR-125b-5p could inhibit the expression of PI3K and AKT gene expression. Fig. 7 miR-125b-5p regulated PI3K/Akt signaling pawthay ( A ) The mRNA levels of PI3K/AKT after transfect with miR-125b-5p mimic/inhibitor in HESCs. (* p  < 0.05, ** p  < 0.01) ( B ) The PI3K/AKT proteins levels after transfect with miR-125b-5p mimic/inhibitor in HESCs by western blot. ( C ) The image analysis of proteins levels. (# p  < 0.05 compared to the inhibitor-NC group; * p  < 0.05 compared to the mimic-NC group) miR-125b-5p regulated PI3K/Akt signaling pawthay ( A ) The mRNA levels of PI3K/AKT after transfect with miR-125b-5p mimic/inhibitor in HESCs. (* p  < 0.05, ** p  < 0.01) ( B ) The PI3K/AKT proteins levels after transfect with miR-125b-5p mimic/inhibitor in HESCs by western blot. ( C ) The image analysis of proteins levels. (# p  < 0.05 compared to the inhibitor-NC group; * p  < 0.05 compared to the mimic-NC group)

Our research demonstrated that miR-125b-5p, miR-125a-5p, miR-30e-3p were significantly decreased, whereas miR-3141, miR-223-3p, miR-142-5p, miR-1246 were significantly increased in patients with EMs. Exosomal miR-125b-5p inhibited VEGF expression and subsequently suppresses the PI3K/Akt signaling pathway, thereby reducing angiogenesis and endometrial stromal cell proliferation, migration, and invasion—key processes in endometriosis pathogenesis.

Endometriosis has been described as a cancer-like process, and tumor derived exosomes have been shown to promote cance cell metastasis by modified the microenvironment of the recipient tissue, making it more suitable for the growth of tumor cells [ 11 ]. Exosomes induced a pro-tumoral microenvironment that promotes tumor progression and survival by promoting angiogenesis, thrombosis, and remodeling of the extracellular matrix [ 12 ]. Furthermore, exosomal miRNAs were also correlated with cancer, such as retinoblastoma, lung cancer, ovarian cancer [ 5 , 13 , 14 ].Ovary is the most important implantation site for ectopic endometrium, and ovarian endometriosis is the subtype most prone to malignant transformation, known as endometriosis-related ovarian cancer [ 15 ]. To explore the relationship between exosomes and endometriosis pathogenesis, many studies had extracted exosomes from plasma, endometriotic stromal cells cultures medium, endometrial tissue and peritoneal fluid. However, these sources may not precisely reflect the biological changes in EMs pathogenesis because many circulating miRNAs may passively released from apoptotic and necrotic cells [ 16 ]. Here, we extracted exosomes from the cyst fluid and pelvic fluid of EMs patients, and sequenced their miRNA profiles. Cyst fluid from EMs patients directly reflects the EMs lesion microenvironment and related pathophysiological processes [ 8 ]. Therefore, cyst fluid-derived exosomal miRNAs may be more directly involved in EMs development. Neuroangiogenesis plays a critical role in the formation and maintenance of endometriotic lesions [ 17 ], which require vascularization to anchor and survive [ 18 ]. Importantly, blood vessels require neural innervation to regulate vasodilation and constriction. Previous studies have demostrated that the endometriotic lesions are highly vascularized and richly innervated [ 19 , 20 ]. Moreover, exosomes derived from endometriotic stromal cells have been shown to enhance angiogenesis, promote neurite outgrowth or transporting neurotransmitters in vitro [ 7 , 21 ]. Consistent with these findings, our GO and KEGG enrichment analyses revealed that differentially expressed exosomal miRNAs from cyst fluid were strongly associated with neurological pathways and blood vessel formation, including regulation of excitatory postsynaptic signaling, neuron migration, axon guidance, and vascular remodeling. We speculate that abnormal cyst fluid-derived exosomes miRNA may promoting endometriosis progression by paracrine or autocrine mechanisms and play an integral part in neuroangiogenesis. From small RNA sequencing, we selected eight candidate exosomal miRNAs (miR-125b-5p, miR-328-3p, miR-125a-5p, miR-30e-3p, miR-3141,miR-223-3p, miR-142-5p, miR-1246.). Among these, miR-125b-5p, miR-328-3p, miR-125a-5p, miR-30e-3p were downregulated, whereas miR-3141, miR-223-3p, miR-142-5p, miR-1246 were upregulated in EMs, with all showing significant differences compared to controls except miR-328-3p. Importantly, the expression levels of these eight exosomal miRNAs from serum sample were consistent with those observed in cyst fluid. Consistent with the findings in cyst fluid, miR-125b-5p levels were significantly lower in the serum of EMs patients compared to the non-EMs control group. Furthermore, ROC analysis identified that miR-125b-5p as the most valuable miRNA for distinguishing EMs (AUC = 0.925; 95% CI, 0.85 ~ 1), indicating a strong association with disease occurrence. Interestingly, we observed no significant downregulation of miR-125b-5p in the peritoneal fluid. This discrepancy compared to cyst fluid and serum may be due to the dilution of lesion-derived exosomes by the physiological peritoneal fluid produced by the extensive normal peritoneal surface. Last but not least, the consistency between cyst fluid and serum profiles supports the utility of circulating miR-125b-5p as a specific biomarker for endometriosis. Notably, compared to traditional biomarkers such as CA125, which is widely used in clinical practice but exhibits limited specificity due to elevation in multiple benign gynecological conditions, miR-125b-5p demonstrated superior diagnostic potential with higher specificity for EMs. Vascular endothelial growth factor (VEGF) and the PI3K/Akt signaling pathway are critical drivers of angiogenesis and cell survival, which are essential for the establishment and maintenance of ectopic endometrial lesions. Dual luciferase reporter assay demonstrated miR-125b-5p mimic significantly decreased VEGF mRNA levels. Elevated VEGF have long been associated with EMs [ 22 ]. We observed miR-125b-5p was markedly downregulated in EMs cyst effusion, which corresponded with increased VEGF expression and led to EMs eventually. These results established a direct regulatory relationship between miR-125b-5p on VEGF and indicate that reduced miR-125b-5p expression contributes to enhanced VEGF signaling in EMs. Overall, these data suggest that miR-125b-5p may serve as a potential non-invasive biomarker candidate, which warrants further validation in larger cohorts. Phosphatidylinositol-3-kinase (PI3K) is an important downstream target of VEGF/VEGFR signaling pathway. In recent years, many studies have demonstrated PI3K/ AKT/ mTOR signaling pathway had plays an important role in the occurrence of breast cancer, lung cancer and prostate cancer and it is closely related to biological behaviors such as cell survival, proliferation and apoptosis [ 23 ]. The VEGF/PI3K/AKT signaling pathways is also essential for regulating the growth, proliferation, migration and angiogenesis of tumor cells, as well as vascular endothelial cells function [ 24 ]. Tu et al. found miRNA-497 had regulated PI3K/Akt signaling pathways throught VEGF to anti-angiogenic [ 25 ]. In addition, VEGF activates Akt/PI3K signaling pathways to promote endothelial cell proliferation, migration, invasion [ 26 ]. Our study demonstrated that miR-125b-5p directly targets VEGF and suppresses the PI3K/Akt signaling pathway, thereby promoting the proliferation and neovascularization of human endometrial cells. These findings indicate that miR-125b-5p promots the proliferation, migration and invasion of human endometrial stromal cells and inhibit cell apoptosis by inhibiting VEGF expression and activating PI3K/AKT signaling pathway. Given that the VEGF/PI3K/AKT pathway involves several molecules, the specific regulatory mechanisms still need to be further studied. Beyond its regulation of angiogenesis and proliferation via the VEGF/PI3K/Akt axis, miR-125b-5p may also play a complex role in the inflammatory and fibrotic microenvironment of endometriosis. Recent evidence suggests that miR-125b-5p can function as an anti-fibrotic role. For example, in pulmonary fibrosis, it alleviates disease progression by targeting BAK1 to inhibit TGF-β1-mediated epithelial-mesenchymal transition (EMT) [ 27 ]. Similarly, in the treatment of intrauterine adhesions, mesenchymal stem cell-derived exosomal miR-125b-5p has been shown to target Smad2 and Smad3, thereby downregulating the TGF-β/Smad signaling pathway and reversing endometrial fibrosis [ 28 ]. In our study, miR-125b-5p was significantly downregulated in the cyst fluid of EMs patients. This downregulation mirrors the fibrotic profiles seen in pulmonary and intrauterine pathologies, suggesting that in endometriosis, miR-125b-5p could imply a loss of protective, anti-fibrotic, or anti-inflammatory regulation though TGF-β/Smad pathway, which may not only unleash VEGF-mediated angiogenesis but also promote the fibrotic adhesions characteristic of endometriosis. Future studies should investigate whether restoring miR-125b-5p levels can simultaneously target the VEGF and TGF-β/Smad signaling networks to stop both lesion growth and fibrosis. Currently, no effective treatments are available that can cure endometriosis or consistently provide long-term symptom remission. Our research offers new mechanistic insights into the pathogenesis of endometriosis. Nevertheless, several limitations should be acknowledged. First, the sample size was relatively small and derived from a single center, potentially introducing selection bias and limiting the generalizability of our findings. Second, the experimental models primarily relied on in vitro analyses, which may not fully recapitulate the complex in vivo microenvironment of EMs. Next, the diagnostic performance of miR-125b-5p has not yet been validated in large, independent cohorts. Future studies should focus on validating these findings across multi-center cohorts with larger and more diverse populations, as well as exploring the translational potential of exosomal miRNAs. For instance, engineered exosome-based delivery of miR-125b-5p mimics or inhibitors may represent a novel therapeutic strategy to modulate VEGF/PI3K/Akt signaling and inhibit EMs progression. Additionally, combining miR-125b-5p with existing biomarkers (e.g., CA125) may further enhance diagnostic accuracy and clinical utility.

Endometriosis (EMs) is a chronic gynecologic disease characterized by the presence of endometrial-like tissue outside the uterus cavity, most commonly in the pelvic cavity. It affects approximately 10% of reproductive-aged women, and its morbidity had continued to increase [ 1 , 2 ].This condition leads to debilitating symptoms such as chronic pelvic pain, dysmenorrhea, and subfertility, significantly impairing quality of life. Despite its clinical prevalence, the pathogenesis of EMs remains incompletely understood. Current theories include retrograde menstruation, coelomic metaplasia, and vascular/lymphatic dissemination [ 3 ]. A major challenge in EMs management is the lack of reliable non-invasive diagnostic biomarkers and targeted therapeutic strategies. In recent years, extensive research has focued on exosomes in human diseases, including cancer, cardiovascular disease, diabetes and reproducive disorders. Exosomes are nano-sized (30–150 nm) extracellular vesicles, which are isolated from varieties biological fluids including blood, saliva, urine, nasal secretions, breast milk, and cerebrospinal fluid [ 4 ]. Exosomes function as intercellular communicators by transporting biologically active proteins, lipids, and nucleic acids (including mRNA, microRNA, and DNA) [ 5 ]. Several studies have investigated the role of exosomes and exosomal miRNAs in endometriosis. MiRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression by binding to messenger RNA (mRNA) targets, thereby inhibiting translation or inducing mRNA degradation. Kasra and colleagues found EMs patients carry unique miRNAs signatures that reflect disease pathophysiology [ 6 ]. Harp et al. demonstrated exosomal miRNAs could contribute to EMs angiogenesis [ 7 ]. Swati et al. identified 42 dysregulated miRNAs in EMs [ 8 ]. Zhang et al. reported that exosomal miRNA-138 could promote EMs through inflammation and apoptosis [ 9 ], while serum exosomal miR-22-3p and miR-320a were significantly higher in EMs patients, suggesting that circulating miRNAs have potential as EMs biomarkers [ 10 ]. These findings highlight the important role of exosomal miRNAs in EMs progression. However, the exosomes analyzed in these studies were mainly derived from plasma, endometrial tissue and peritoneal fluid, which may not fully represent the actual pathological microenvironment of EMs. Ovarian cyst was the one of clinical symptoms for stage II- Ⅲ endometriosis. Therefore, exosome extracted from ovarian cyst fluid may better reflect the EMs microenvironment. Analyzing cyst fluid-derived exosomes may provide deeper insight into the pathological mechanisms of EMs. Vascular endothelial growth factor (VEGF) and the PI3K/Akt signaling pathway are critical drivers of angiogenesis and cell survival, which are essential for the establishment and maintenance of ectopic endometrial lesions. In this study, we aimed to characterize exosomes derived from cyst fluid of EMs patients, identify differentially expressed miRNAs, and investigate their functional roles in endometrial cell biology. Specifically, we focused on miR-125b-5p, a significantly downregulated miRNA in EM-derived exosomes, to explore its effects on cell proliferation, migration, and invasion, as well as its molecular mechanisms involving VEGF and the PI3K/Akt pathway. By linking exosomal miRNA dysregulation to key pathogenic processes in EMs, this study seeks to identify novel diagnostic biomarkers and therapeutic targets for EMs.

Below is the link to the electronic supplementary material. Supplementary Material 1 Supplementary Material 1 Supplementary Material 2 Supplementary Material 2 Supplementary Material 3 Supplementary Material 3