Overexpression of miR-21-5p as a potential pathogenesis marker for ovarian endometriosis

Background Endometriosis is a benign gynecological disease characterized by the growth of endometrial cells beyond the uterus, forming endometriotic cyst tissues called ovarian endometriomas. MicroRNAs (miRNAs) are small, non-coding RNAs that epigenetically control the physiological and pathological processes of different diseases, including endometriosis. In this study, we screened the expression levels of 11-selected miRNAs, namely miR-21-5p, miR-200c-3p, miR-19a-3p, miR-203-3p, miR-181b-5p, miR-182-5p, miR-let7a-5p, miR-205-5p, miR-200b-3p, miR-16-5p, and miR-222-3p in ovarian endometriomas relative to eutopic endometrial tissues using quantitative real-time PCR (qPCR). In addition, the level of mRNA expression of lumican (LUM), an extracellular matrix proteoglycan (PG), and a putative target of miR-21-5p was quantified by qPCR.

Our screening qPCR results showed that 9 miRNAs were upregulated (miR-21-5p, miR-200c-3p, miR- 19a-3p, miR-203-3p, miR-181b-5p, miR-182-5p, miR-let7a-5p, miR-205-5p, and miR-200b-3p), whereas 2 miRNAs were downregulated (miR-16-5p and miR-222-3p) in ovarian endometriomas compared to eutopic endometrium. A significant overexpression of miR-21-5p in ovarian endometrioma was further independently verified by qPCR. Using bioinformatics tools, Gene Ontology Kyoto Encyclopedia of Genes and Genomes pathways, and protein– protein interactions, we identified differentially expressed genes and several pathways regulated by miR-21-5p that may contribute to endometriosis progression. Among them, LUM was found to be significantly diminished expressed in ovarian endometriomas compared to eutopic endometrium.

In conclusion, this study identified miR-21-5p and LUM as potential factors that may contribute to ovarian endometriomas’ pathogenesis.

Endometriosis, Ovarian endometriomas, microRNA, miR-21, Lumican Open Access © The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Journal of Basic and Applied Zoology †Mohamed Mahmoud, Amr Ahmed WalyEldeen, Sherif Abdelaziz Ibrahim, and Hebatallah Hassan contributed equally to this work. *Correspondence: Sherif Abdelaziz Ibrahim [email protected]; [email protected] Hebatallah Hassan [email protected] 1 Department of Zoology, Faculty of Science, Cairo University, Giza 12613, Egypt 2 Department of Gynecology and Obstetrics, Faculty of Medicine, Al- Azhar University, Cairo 11651, Egypt 3 Department of Gynecology and Obstetrics, Faculty of Medicine, Benha University, Banha 13518, Egypt 4 Consultant of Obstetrics and Gynecology and Minimally Invasive Surgery, Omam Comprehensive Endometriosis Centre, Giza 22311, Egypt 5 Pathology Department, Faculty of Medicine, Aswan University, Aswan 81528, Egypt Page 2 of 15Mahmoud et al. The Journal of Basic and Applied Zoology (2025) 86:1

Endometriosis is a gynecological, genetic, and estrogen- dependent autoimmune disorder in which endometrial cells grow outside the uterus, primarily in the ovaries and peritoneum (Chen et al., 2023; Nisolle & Donnez, 1997; Shao et  al., 2014). Ovarian endometriomas can severely impair ovary reserve and may lead to infertility due to laparoscopic ovarian cystectomies (Bulun, 2019; Gao et  al., 2023; Kitajima et  al., 2011; Yılmaz Hanege et  al., 2019). Other clinical complications, besides infertility, are substantially associated with endometriosis, such as chronic pelvic pain, dyspareunia, and dysmenorrhea. Consequently, decreased quality of life and economic burden associated with endometriosis therapy repre - sent the major challenges for this disease (Rogers et  al., 2013). Although multiple efforts have been dedicated to discovering the cause of endometriosis, the fundamental molecular mechanisms underlying the cause of 10% of women harboring endometriosis have not yet been dis - covered (Hu et al., 2022). The ovarian endometriotic cyst is commonly referred to as a chocolate cyst as it is filled with menstruation-like hemorrhagic blood, giving it a dark brown appearance. The accumulation of this hem - orrhagic blood inside the cyst prompts the recruitment of the macrophages, which in turn phagocytize and lyse the blood cells, releasing heme and iron, and inducing the secretion of pro-inflammatory cytokines (Guo et al., 2015; Simoni et  al., 1994; Wyatt et  al., 2023). Because of prolonged exposure to inflammation, cysts begin to develop fibrosis (Houghton & McCluggage, 2011). Intriguingly, it has been reported that many ovarian endometriotic cysts may be originated from the meta - plastic fallopian tube instead of eutopic endometrium since the specific markers of fallopian tube may be seen in ovarian endometriotic lesions, which in turn explains the unresponsive to hormonal treatments in many cases (Hill et al., 2020; Wang et al., 2023; Yuan et al., 2014). MicroRNAs (miRNAs) are crucial epigenetic regula - tors of genes related to the formation and progression of endometriotic lesions; consequently, they appear to be potentially attractive candidates (Hawkins et  al., 2011). miRNAs can suppress the transcription or translation when it binds to the 3′ untranslated region (UTR) of the targeted mRNA (Lee et al., 1993; Pasquinelli et al., 2000). miRNAs have been previously demonstrated to regulate the hallmarks of cancer, including proliferation, death, angiogenesis, differentiation, and migration (Hu et  al., 2022; Zhang et  al., 2019). A previous microarray study revealed aberrant expression of miRNA in ectopic endo - metrium compared to eutopic endometrium (Teague et  al., 2009). However, specific miRNA expression pat - terns in women with ovarian endometriomas have not yet been fully explored. So, this study aimed to screen 11-selected dysregulated miRNA expression patterns and identify putative targets, which may have a potential role in ovarian endometriomas pathogenesis.

Sample collection This study was initiated after obtaining approval from the institutional review board (IRB) at the Faculty of Medicine, Al-Azhar University (protocol number: 0000195) and the local Ethics Committee of the Fac - ulty of Medicine at Aswan University (Protocol No. Asw.U./677/10/22). Aligned with the regulations of the Declaration of Helsinki, all patients with ovarian endo - metriosis enrolled in this study signed consent form to participate. Females aged 24–49  years diagnosed with ovarian endometriomas through laparoscopy and were not on hormone replacement treatment for at least a year were recruited for this study. Ovarian endometri - otic cyst tissues (n = 14) and eutopic endometrial tissues (n = 8) were collected. The patients with chronic autoim - mune disease, infectious conditions, acute inflammation, or cancer were excluded. For the RNA extraction, the tissue samples were snap-frozen in liquid nitrogen and subsequently stored at − 80  °C until needed for further analysis. Histological examination Endometrium and ovarian endometriomas were fixed in neutral-buffered formalin (10%), followed by dehydration steps through ascending ethanol series. Tissue sections were treated with xylene to eliminate the alcohol, and paraffin was used to embed the tissue. Hematoxylin and eosin were used to stain tissue sections as previously described (Fischer et  al., 2008; Velho et  al., 2023) with a thickness of 5 μm, and a pathologist examined the sections in a blind manner. RNA isolation and cDNA synthesis The miRNeasy Mini Kit (cat. no.217004, Qiagen, Hilden, Germany) was used to extract total RNA from eutopic endometrium and ovarian endometrioma for miRNA and mRNA expression analysis. Tissues were first homogenized in QIAzol reagent (Qiagen) and incubated with 200 µL chloroform to obtain the RNA’s aqueous phase. Subsequently, ethanol-treated RNA was transferred to the RNeasy mini-column, according to the manufacturer’s instructions. The concentration and purity of the eluted RNA were measured at an absorb - ance of 260 and 280 nm using an Infinite ®200 PRO NanoQuant (Tecan, Zürich, Switzerland). Using the miScript II RT kit (catalog no. 218160, Qiagen), 1 μg of RNA was reverse-transcribed into cDNA encoding miRNAs in reverse-transcription reaction components Page 3 of 15 Mahmoud et al. The Journal of Basic and Applied Zoology (2025) 86:1 of 5X hi-spec buffer, 10 × miScript Nucleics Mix, and miScript reverse transcriptase mix. The reaction was incubated for 60 min at 37 °C in Veriti ™ 96-Well Ther- mal Cycler (Applied Biosystem, CA, USA) followed by 5  min at 95  °C to inactivate miScript reverse tran - scriptase mix. To determine the mRNA expression level of the LUM gene, the RevertAid First Strand cDNA Kit (cat. no. k1622, Thermo Scientific, Vilnius, Lithuania) was used to reverse transcribe total RNA (1 μg) into cDNA as we described before (Fahim et al., 2020). Quantitative real‑time PCR The relative miRNA expression levels were quanti - fied using the miScript SYBR Green PCR Kit (cat. no.218073, Qiagen) and miScript Primer Assays (Qiagen) for 11-selected miRNAs: hsa-miR-21-5p (MS00009079), hsa-miR-200c-3p (MS00003752), hsa-miR-19a-3p (MS00003192), hsa-miR-203-3p (MS00003766), hsa-miR-181b-5p (MS00006699), hsa-miR-182-5p (MS00008855), hsa-miR-let7a- 5p (MS00031220), hsa-miR-205-5p (MS00003780), hsa-miR-200b-3p (MS00009016), hsa-miR-16-5p (MS00031493), and hsa-miR-222-3p (MS00007609). For the screening of 11-selected miRNA expression lev - els, an equal concentration of the isolated RNA (ovar - ian endometriomas, n = 10 and eutopic endometrial tissues, n = 8) was pooled and reverse transcribed into cDNA. For the validation step, RNA isolated from ovar - ian endometriomas (n = 14) and eutopic endometrial tissues (n = 8) was individually reverse transcribed into cDNA. Data were normalized to the endogenous ribo - nuclear RNA RNU6-2-11 (MS00033740). The cycling conditions used for miRNAs were as follows: 15  min initial activation at 95 °C step, then 40 cycles consisting of 15 s at 94 °C, 30 s at 55 °C, and 30 s at 70 °C. The rela - tive mRNA expression levels of LUM  normalized to the endogenous housekeeping gene β-actin (ACTB ) were assessed using Maxima SYBR Green qPCR Master Mix (2X) (cat. No. k1061, Thermo Scientific, Vilnius, Lithu - ania) in StepOnePlus ™ Real-Time PCR System (Applied Biosystems). The cycling conditions used for mRNAs are as follows: A 10 min-initial activation step at 95 °C, then 40 cycles consisting of 15 s at 95 °C, and 1 min at 60 °C. The 2−ΔΔCt method was used to express the fold change of miRNA and mRNA expression levels as we previously described (Fahim et  al., 2020). The primer sequences used for LUM were forward primers 5′ -AAC ATA CCA ACT GTC AAT GAA AAC C-3′, reverse prim - ers 5′ -TGC CAT CCA AAC GCA AAT GCTTG-3′ and for ACTB were forward primer 5 ′-CAC CAT TGG CAA TGA GCG GTTC-3′ and reverse primer 5′ -AGG TCT TTG CGG ATG TCC ACGT-3′. In‑silico analysis of miRNA target genes and pathways exploration To find the possible targets of the dysregulated miRNA, we utilized the miRbase (miRDB) (http:// mirdb. org/ index. html) (accessed on August 1, 2024) (Pathan et al., 2015, 2017). The predicted target genes (469 genes) were used to analyze the gene ontology (GO) function and the Kyoto Encyclopedia of Genes and Genomes (KEGG) using the online Database for Annotation, Visualization, and Integrated Discovery (DAVID) software (https:// david. ncifc rf. gov/ home. jsp) (accessed on August 1, 2024) using FDR < 0.05 (Huang et al., 2009a, 2009b). Finally, the web platform STRING (https:// string- db. org) (accessed on August 1, 2024) integrated the protein–protein inter - action (PPI) network and identified key candidate genes (Hub Genes) and pathways (Szklarczyk et al., 2019). Enrichr database (Chen et  al. 2013; Xie et  al., 2021a) (https:// maaya nlab. cloud/ Enric hr/) (accessed on Octo - ber 10, 2024) was used to identify the biological pathways associated with LUM, ST3GAL6, C7, LRRC57, PTPRG, MATN2, ADAMTS3, FASLG, TLR4, ANXA1, TGFB2, TIMP3, S100A10, COL4A1, and LAMA4 representing genes that are connected with LUM within the miR- 21-5p target genes. This web-based software contains several gene set libraries, among which we used the Else - vier Pathway Collection analyses (Chen et al., 2013; Kule- shov et al., 2016; Xie et al., 2021b). Statistical analysis The Statistical Package for Social Science (SPSS) version 25 was used to analyze data. The normally distributed data were evaluated using parametric tests, and a nor - mality test was performed using skewness and kurtosis. Using the Student’s T-test, the statistical significance lev- els for the difference between the two groups were used. Using Pearson’s correlation, the correlation between the two variables was evaluated. A p-value < 0.05 was con - sidered significant for all tests, and data are presented as mean ± SEM. GraphPad Prism 8 was used to generate graphs.

Histopathological examination of the endometria and endometriotic cysts Biopsies of eutopic endometrium and ovarian endometriotic cysts were obtained. Eutopic endometrium samples exhibit typical morphological characteristics of the endometrium (Franco- Murillo et  al., 2015), comprising tubular glands and endometrial stroma that are associated with blood vessels and immune cells (Yamaguchi et  al., 2021). Ovarian endometriomas specimens were histologically Page 4 of 15Mahmoud et al. The Journal of Basic and Applied Zoology (2025) 86:1 confirmed by the presence of glands resembling the endometrium, endometrial-like stroma, and an aggregation of “hemosiderin-containing macrophages” that is considered a unique histological feature of the endometriotic cyst (Fig.  1). Of note, two of the three histological features of endometriosis are sufficient for diagnosing the patient with endometriosis (Houghton & McCluggage, 2011). Distinct miRNAs expression pattern between ovarian endometriomas tissues compared to eutopic endometria tissues For screening dysregulated 11-selected miRNAs, we performed qPCR for cDNA from pooled samples of 10 ovarian endometriomas and 8 eutopic endometrial tissue samples. Our qPCR data, as shown in Fig.  2, revealed that miR-21-5p, miR-200c-3p, miR-19a-3p, miR-203a-3p, miR-181b-5p, miR-182-5p, miR- let-7a-5p, miR-205-5p, and miR-200b-3p were upregulated, whereas miR-16-5p and miR-222-3p were downregulated in ovarian endometriomas compared to eutopic endometria as depicted in Table 1 . Upregulation of miR‑21‑5p in ovarian endometriomas Since miR-21-5p, miR-let7a-5p, and miR-200b-3p were the top upregulated miRNAs in pooled samples of ovarian endometriomas relative to eutopic endometria, we verified the difference in their expression individually. Interestingly, miR-21-5p was significantly upregulated (twofold change, P 0.05) (Fig. 3B), and miR-200b-3p was upregulated (threefold change, P > 0.05) in ovarian endometriomas relative to eutopic endometrial tissue. However, their expression did not reach a significant level (Fig. 3 C). Prediction of miR‑21‑5p target genes, Gene ontology function, and KEGG pathway analysis Using the miRDB online database, we identified LUM as a potential target for miR-21-5p. qPCR

demonstrated a significant downregulation of LUM expression (by − 0.75-fold, P < 0.05) in ovarian endometriomas compared to eutopic endometria (Fig.  4A). A positive correlation was observed between Fig. 1 Histopathological sections of the normal endometria and ovarian endometriotic cysts. A Normal endometria proliferative type showing endometrial glands (red arrow), stroma (blue arrow), and blood vessels (green arrow). B Sections of an endometriotic cyst of the ovary showing endometrial-type epithelium (red arrow), endometrial-type stroma (blue arrow), and hemosiderin-laden macrophages (green arrow). Magnification: 400X Fig. 2 Differentially expressed miRNAs in ovarian endometriomas compared to eutopic endometrial tissues. The bar graph shows the log2 fold change in expression levels of 11-selected miRNAs in ovarian endometriomas tissues (pooled n = 10) and eutopic endometrial tissues (pooled n = 8). miR-21-5p, miR-200c-3p, miR-19a-3p, miR-203a-3p, miR-181b-5p, miR-182-5p, miR-let-7a-5p, miR-205-5p, and miR-200b-3p were upregulated, while miR-16-5p and miR-222-3p were downregulated in ovarian endometriomas relative to eutopic endometrial tissues Table 1 The fold change of differentially expressed miRNAs in ovarian endometriomas compared to eutopic endometrial tissue miRNAs Log2 fold change (2^‑ ΔΔCT) Upregulated miR-200b-3p 8.40 miR-let-7a-5p 6.93 miR-21-5p 5.08 miR-200c-3p 4.42 miR-19a-3p 3.37 miR-203-3p 2.87 miR-205-5p 2.34 miR-181b-5p 1.97 miR-182-5p 1.92 Downregulated miR-16-5p − 1.11 miR-222-3p − 5.00 Page 5 of 15 Mahmoud et al. The Journal of Basic and Applied Zoology (2025) 86:1 miR-21-5p and LUM in ovarian endometriomas tissue, while a negative correlation was found in eutopic endometrium. However, neither correlation reached statistical significance (P > 0.05, Fig.  4B). This suggests that miR-21-5p may regulate the expression of LUM on a posttranscriptional and/or translational level, but this speculation should be verified in a future mechanistic study. Subsequently, we retrieved 469 target genes for miR- 21-5p from the miRbase database (miRDB) and performed a comprehensive analysis using the DAVID tool to explore gene ontology and KEGG pathways. The gene ontology analysis, namely biological processes, cellular components, and molecular functions linked to the identified target genes for miR-21-5p was performed. Notably, the biological processes included regulation of transcription, chromatin, and signal transduction, with key cellular components encompassing nucleus, cytosol, cytoplasm, and nucleoplasm. The molecular function category exhibited enrichment in protein, DNA, metal ion, zinc ion binding, protein serine/threonine kinase activity, and sequence- specific DNA interaction. All other functional annotations related to the targets of miR-21-5p are depicted in Table 2. Moreover, the KEGG analysis uncovered several path - ways with a potential to contribute to ovarian endometrio- sis, including MAPK, P13K-Akt, RAS, miRNAs in cancer, proteoglycans in cancer, polycomb repressive, FoxO, TGF- beta, EGFR, prolactin, and Hippo signaling pathways sign- aling pathways as depicted in Table 3. Fig. 3 qPCR for the expression of miR-21-5p, miR-let7a-5p, and miR-200b-3p in ovarian endometriomas and eutopic endometria. The relative miRNA expression levels of miR-21-5p (A), miR-let7a-5p (B), and miR-200b-3p (C) in ovarian endometriomas (n = 10–14) compared to eutopic endometrial tissue (n = 8). Bars represent means ± SEM. *P < 0.05 as determined by Student’s t-test Fig. 4 A The mRNA expression levels of the LUM assessed by qPCR in ovarian endometriomas (n = 14) compared to eutopic endometria tissue (n = 8). Bars represent means ± SEM. *P < 0.05 as determined by Student’s t-test. B Pearson’s correlation between miR-21–2-5p and LUM level in eutopic endometria (n = 8) and ovarian endometriomas tissues (n = 14) Page 6 of 15Mahmoud et al. The Journal of Basic and Applied Zoology (2025) 86:1 Protein–protein interaction network of predicted target genes of miR‑21‑5p We employed the String database to explore potential protein–protein interaction networks among the predicted target genes (https:// string- db. org/), including 469 proteins identified as miR-21-5p targets. The resulting network unveiled numerous interactions among these proteins, forming three distinct clusters. The first cluster, comprising 173 proteins, is associated with molecular function (specifically Glycogen binding), KEGG pathways (such as Cytokine-Cytokine receptor interaction), and subcellular localization in the extracellular region. The second cluster, consisting of 140 proteins, is linked to pathways involving Ras, NTRK2 (TRKB), and regulation of the microtubule cytoskeleton. The third cluster of 153 proteins is associated with various pathways, including notch, TGF-beta, p53, androgen, and prolactin. This comprehensive analysis of protein– protein interaction networks sheds light on the intricate relationships and functional implications of miR-21-5p target proteins, providing valuable insights into their roles in different biological processes and pathways (Fig. 5). Within the PPI network, LUM was found to be directly connected with several proteins, including LUM, ST3GAL6, C7, LRRC57, PTPRG, MATN2, ADAMTS3, FASLG, TLR4, ANXA1, TGFB2, TIMP3, S100A10, COL4A1, and LAMA4 indicating potential interactions that could influence its biological role and involvement in various signalling pathways (Fig.  6A). In addition, Enrichr database (https:// maaya nlab. cloud/ Enric hr/) was employed to elucidate the functional pathways associated with LUM, ST3GAL6, C7, LRRC57, PTPRG, MATN2, ADAMTS3, FASLG, TLR4, ANXA1, TGFB2, TIMP3, S100A10, COL4A1, and LAMA4 representing genes that are connected with LUM within the miR-21-5p target genes. The functional enrichment analysis based on Elsevier Pathway Collection analyses revealed that these proteins are involved in endometriosis (Fig. 6B).

In our study, we screened for 11-selected dysregulated miRNA expressions and their potential targets, which may be involved in the pathogenesis of endometriosis. Our qPCR results revealed the overexpression of different miRNAs, namely, miR-21-5p, miR-200c-3p, miR-19a-3p, miR-203-3p, miR-181b-5p, miR-182-5p, miR-let7a-5p, and miR-200b-3p, whereas two miRNAs, miR-16-5p and miR-222-3p, were downregulated in ovarian endometriomas compared to eutopic endometrial tissues. We further verified a significant overexpression of miR-21-5p in endometriomas compared to eutopic endometria tissues. Different studies reported miRNA signatures in endometriotic lesions and indicated altered levels of miR-1, miR-29c, miR-34c, miR-141, miR-183, miR-196b, miR-145, miR- 200a, miR-200b, miR-200c, miR-202, miR-100, miR-365, and miR-375 (Shantanam & Mueller, 2018). Several of these miRNAs are also known to regulate angiogenesis, cell proliferation, invasion, and cell adhesion, in addition to epithelial-mesenchymal transition (EMT), unique hallmarks associated with endometriosis (Braicu et  al., 2017; Saare et  al., 2017). Another study underscored another set of dysregulated miRNAs such as miR-20a, miR-15, miR-29c, miR-23a/b, miR-126, miR-142, miR- 145, miR-183, miR-199a, and miR–451 in endometriotic lesions (Nothnick, 2017). These dysregulated miRNAs may act as “drivers” that contributed to events of endometriosis pathophysiology or as “passengers” that were altered due to disease pathogenesis (Nothnick, 2017). miR-21 governs biological processes such as cell proliferation, invasion, and angiogenesis (Krichevsky & Gabriely, 2009). These processes are crucial for the establishment and progression of endometriosis. In this context, our finding of elevated miR-21-5p expression further supports its role in ovarian endometriosis and agrees with different studies. For example, a very recent study reported the role played by miR-21-5p in ovarian endometrial cysts, where the exosomal miR- 21-5p derived from endometrial stromal cells augments proliferation, migration, and angiogenesis of human umbilical vein endothelial cells (HUVECs) via targeting TIMP3 (Sun et al., 2024). miR-21-5p is the second most upregulated miRNA in women with endometriosis (Braza-Boïls et  al., 2014) and in endometrial epithelial tissue of porcine and mouse models during embryo implantation, and its inhibition impedes proliferation and migration of endometrial cells, via programmed cell death 4 (PDCD4) (Hua et  al., 2020). Suppression of miR-21 expression in endometrial stromal cells (HESCs) isolated from patients undergoing laparoscopic surgery induces apoptotic cell death via caspase-3 overexpression (Park et  al., 2018). Another mechanistic study reported that overexpression of long non-coding RNA (lnRNA) MEG3 inhibits endometrial cell proliferation and invasion via downregulation of miR-21-5p (Yang et  al., 2023), supporting our hypothesis that miR-21-5p may contribute to the pathogenesis of endometriomas. Interestingly, a study reported that oncomiR-21 acts as a circulating biomarker for mild and severe forms of endometriosis (Rozati et  al., 2023), implying its useful - ness as a future non-invasive biomarker for diagnosis of patients with ovarian endometriosis. Epithelial-mesenchymal transition (EMT) program plays a central role in endometriosis pathogenesis (Szymański et  al., 2024). A higher expression of miR-21 and lower expression of EMT markers TGF-β1, SMAD3, Page 7 of 15 Mahmoud et al. The Journal of Basic and Applied Zoology (2025) 86:1 Table 2 Gene ontology analysis of miR-21-5p target genes Category Term Count % P‑value Biological process Negative regulation of transcription by RNA polymerase II 54 11.6 0.00000 Positive regulation of transcription by RNA polymerase II 60 12.9 0.00000 Negative regulation of stem cell differentiation 6 1.3 0.00004 Proteasome-mediated ubiquitin-dependent protein catabolic process 15 3.2 0.00150 Regulation of transcription by RNA polymerase II 55 11.8 0.00150 Positive regulation of osteoblast differentiation 8 1.7 0.00190 Hippo signaling 5 1.1 0.00210 Interleukin-6-mediated signaling pathway 4 0.9 0.00330 Roof of mouth development 7 1.5 0.00350 Signal transduction 43 9.2 0.00450 Glycogen metabolic process 5 1.1 0.00530 Ephrin receptor signaling pathway 6 1.3 0.00540 Positive regulation of protein phosphorylation 12 2.6 0.00660 Cellular response to cytokine stimulus 5 1.1 0.00660 Negative regulation of neuron apoptotic process 10 2.1 0.00680 Cellular response to mechanical stimulus 7 1.5 0.00740 Somatic stem cell population maintenance 6 1.3 0.00740 Regulation of angiogenesis 5 1.1 0.00810 Protein transmembrane transport 4 0.9 0.00810 Negative regulation of transforming growth factor beta receptor signaling pathway 9 1.9 0.00830 Positive regulation of MAPK cascade 11 2.4 0.00960 Bud elongation involved in lung branching 3 0.6 0.00960 Positive regulation of epithelial cell migration 5 1.1 0.00980 Ventricular septum morphogenesis 5 1.1 0.00980 Positive regulation of cell population proliferation 21 4.5 0.00980 Cartilage development 6 1.3 0.01400 Hemopoiesis 6 1.3 0.01400 Positive regulation of bone mineralization 5 1.1 0.01400 Intracellular signal transduction 18 3.9 0.01700 Hematopoietic stem cell homeostasis 4 0.9 0.01700 Negative regulation of Ras protein signal transduction 4 0.9 0.01700 Positive regulation of myoblast differentiation 5 1.1 0.01900 Protein K48-linked ubiquitination 7 1.5 0.01900 Nuclear pore complex assembly 3 0.6 0.02000 Regulation of glycogen biosynthetic process 3 0.6 0.02000 Positive regulation of canonical Wnt signaling pathway 8 1.7 0.02000 Lung alveolus development 5 1.1 0.02000 Negative regulation of epithelial to mesenchymal transition 5 1.1 0.02200 mRNA transcription by RNA polymerase II 5 1.1 0.02300 Page 8 of 15Mahmoud et al. The Journal of Basic and Applied Zoology (2025) 86:1 Table 2 (continued) Category Term Count % P‑value Glial cell migration 3 0.6 0.02400 Transforming growth factor beta receptor signaling pathway 7 1.5 0.02400 Negative regulation of translation 7 1.5 0.02400 Negative regulation of BMP signaling pathway 6 1.3 0.02500 Epithelial cell proliferation 5 1.1 0.02500 Negative regulation of cell adhesion 5 1.1 0.02700 Negative regulation of signal transduction 5 1.1 0.02700 Positive regulation of Notch signaling pathway 5 1.1 0.02700 Organ induction 3 0.6 0.02800 Positive regulation of hepatocyte proliferation 3 0.6 0.02800 ERK1 and ERK2 cascade 5 1.1 0.02800 Positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction 10 2.1 0.02800 Positive regulation of DNA-templated transcription 25 5.4 0.03000 Phosphatidylinositol 3-kinase/protein kinase B signal transduction 6 1.3 0.03000 Outflow tract morphogenesis 5 1.1 0.03000 Extrinsic apoptotic signaling pathway 5 1.1 0.03000 Animal organ development 4 0.9 0.03100 Positive regulation of ERK1 and ERK2 cascade 11 2.4 0.03200 Protein import into nucleus 7 1.5 0.03200 Negative regulation of DNA-templated transcription 21 4.5 0.03300 Negative regulation of activin receptor signaling pathway 3 0.6 0.03300 Positive regulation of nuclear-transcribed mRNA poly(A) tail shortening 3 0.6 0.03300 Positive regulation of MAP kinase activity 5 1.1 0.03400 Embryonic forelimb morphogenesis 4 0.9 0.03600 Positive regulation of cardiac muscle cell proliferation 4 0.9 0.03600 Fibroblast growth factor receptor signaling pathway 5 1.1 0.04000 Chondrocyte differentiation 5 1.1 0.04000 Odontogenesis of dentin-containing tooth 5 1.1 0.04200 Regulation of synaptic transmission, glutamatergic 4 0.9 0.04200 Protein phosphorylation 15 3.2 0.04300 Regulation of epithelial to mesenchymal transition 3 0.6 0.04300 Positive regulation of nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay 3 0.6 0.04300 BMP signaling pathway 6 1.3 0.04300 Negative regulation of mesenchymal cell proliferation involved in lung development 2 0.4 0.04400 AMPA selective glutamate receptor signaling pathway 2 0.4 0.04400 Muscle cell fate determination 2 0.4 0.04400 Viral RNA genome replication 2 0.4 0.04400 Hepatic stellate cell activation 2 0.4 0.04400 Regulation of cell migration 7 1.5 0.04500 Page 9 of 15 Mahmoud et al. The Journal of Basic and Applied Zoology (2025) 86:1 Table 2 (continued) Category Term Count % P‑value Regulation of ERK1 and ERK2 cascade 4 0.9 0.04500 Regulation of cell population proliferation 8 1.7 0.04600 Positive regulation of nitric-oxide synthase biosynthetic process 3 0.6 0.04800 Ureteric bud development 4 0.9 0.04900 Oligodendrocyte differentiation 4 0.9 0.04900 Cellular component Nucleoplasm 131 28.1 0.00000 Chromatin 51 10.9 0.00000 Nucleus 177 38 0.00001 Cytosol 161 34.5 0.00004 Cytoplasm 163 35 0.00005 Nuclear matrix 12 2.6 0.00012 Ubiquitin ligase complex 11 2.4 0.00020 Nuclear body 20 4.3 0.00022 Protein-containing complex 30 6.4 0.00046 Golgi apparatus 41 8.8 0.00093 Adherens junction 13 2.8 0.00140 Postsynaptic density 14 3 0.00260 Collagen-containing extracellular matrix 19 4.1 0.00350 SWI/SNF complex 5 1.1 0.00480 Early endosome membrane 12 2.6 0.00550 RNA polymerase II transcription regulator complex 9 1.9 0.00640 Nuclear envelope 11 2.4 0.01700 Transcription regulator complex 12 2.6 0.01800 Axon 16 3.4 0.01900 Actin cytoskeleton 13 2.8 0.02000 Dendritic spine 9 1.9 0.02500 Intracellular membrane-bounded organelle 30 6.4 0.02600 Protein phosphatase type 1 complex 3 0.6 0.02700 BRCA1-A complex 3 0.6 0.02700 Cell cortex 9 1.9 0.02900 Spindle pole 8 1.7 0.03900 Fibrillar center 8 1.7 0.04700 Molecular Function Protein binding 345 74 0.00000 DNA-binding transcription factor activity 30 6.4 0.00002 Ubiquitin-protein transferase activity 16 3.4 0.00026 DNA-binding transcription activator activity, RNA polymerase II-specific 25 5.4 0.00049 Transcription cis-regulatory region binding 16 3.4 0.00055 Glycogen binding 4 0.9 0.00092 Signaling receptor binding 20 4.3 0.00095 DNA-binding transcription factor activity, RNA polymerase II-specific 49 10.5 0.00200 Metal ion binding 88 18.9 0.00210 DNA-binding transcription factor binding 12 2.6 0.00220 Sequence-specific double-stranded DNA binding 25 5.4 0.00280 RNA polymerase II cis-regulatory region sequence-specific DNA binding 45 9.7 0.00500 Signaling adaptor activity 7 1.5 0.00510 Sequence-specific DNA binding 15 3.2 0.00640 Page 10 of 15Mahmoud et al. The Journal of Basic and Applied Zoology (2025) 86:1 and ILK genes are associated with loss of the endometrial epithelial phenotype and EMT in ovarian endometrioma compared to matched eutopic endometrium (Zubrzycka et al., 2023), further supporting the role of miR-21-5p in ovarian endometriosis. Our findings further revealed a significant downregulation of LUM , a putative target of miR-21-5p, in ovarian endometriomas compared to eutopic endometrial tissues. LUM is important in maintaining healthy tissue architecture (Chakravarti, 2002). Additionally, in the tumor biology context, LUM expression is linked to signaling events relevant to pro- or anti-tumorigenic consequences. Most of its pro-tumorigenic actions are shown in stomach, liver, and bladder cancers and are associated with a poorer clinical prognosis. The pro-tumorigenic activities of LUM activate FAK, MAPK, and MMP-9 (Appunni et al., 2021; Chen et al., 2017; De Wit et al., 2017; Hsiao et  al., 2020; Radwanska et  al., 2012). On the contrary, LUM has anticancer actions in breast and pancreatic cancers, as well as in melanoma that are linked to positive clinical outcomes (Appunni et al., 2021; Brezillon et  al., 2009; Brézillon et  al., 2007; Stasiak et  al., 2016; Yang et  al., 2018). The anti-tumor activity of LUM was associated with the reduction of cancer cell invasion and migration. So, increased migration may be associated with decreased LUM expression (Appunni et  al., 2021; Hsiao et  al., 2020; Yang et  al., 2018). In the context of endometriosis, the notion of downregulated LUM in our study suggests that LUM may function similarly to its role in cancer by influencing cellular behaviors, such as invasion and migration. Given its involvement in ECM remodeling, reduced LUM levels could facilitate a more permissive environment for the invasive capacity of ectopic endometrial tissue. Although only a few studies have examined LUM’s role in endometriosis, there are conflicting findings: two studies reported Table 2 (continued) Category Term Count % P‑value Protein serine/threonine kinase activity 18 3.9 0.00730 Growth factor activity 10 2.1 0.01400 Chromatin binding 21 4.5 0.01800 Protein serine kinase activity 16 3.4 0.02200 Single-stranded RNA binding 5 1.1 0.02400 Transcription coregulator activity 8 1.7 0.02600 Zinc ion binding 31 6.7 0.02600 lncRNA binding 4 0.9 0.02800 mRNA 3′-UTR AU-rich region binding 4 0.9 0.02800 Nuclear receptor activity 5 1.1 0.03500 Beta-catenin binding 7 1.5 0.04200 Chromatin DNA binding 6 1.3 0.04800 Ribonucleoprotein complex binding 4 0.9 0.04900 Count: the number of target genes associated with each term in gene ontology category. %: indicates the proportion of target genes associated with each term in gene ontology category. P-value: represents the statistical significance of enrichment for each term. Table 3 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of miR-21-5p Term Count % P‑value MAPK signaling pathway 19 4.1 0.0004 PI3K-Akt signaling pathway 17 3.6 0.015 Ras signaling pathway 15 3.2 0.0019 MicroRNAs in cancer 14 3 0.041 Proteoglycans in cancer 13 2.8 0.0044 Polycomb repressive complex 10 2.1 0.0002 FoxO signaling pathway 10 2.1 0.0047 Hepatitis C 10 2.1 0.015 Hepatitis B 10 2.1 0.018 Amoebiasis 9 1.9 0.0034 TGF-beta signaling pathway 9 1.9 0.0049 Neurotrophin signaling pathway 9 1.9 0.0086 EGFR tyrosine kinase inhibitor resistance 7 1.5 0.013 Insulin resistance 7 1.5 0.049 Prolactin signaling pathway 6 1.3 0.028 Non-small cell lung cancer 6 1.3 0.031 Hippo signaling pathway—multiple species 4 0.9 0.033 Page 11 of 15 Mahmoud et al. The Journal of Basic and Applied Zoology (2025) 86:1 LUM’s upregulation in endometriosis (Irungu et  al., 2019; Sahar et  al., 2021), while another report revealed downregulation of LUM in mural granulosa cells from women with endometriosis (Kedem et  al., 2022). These discrepancies in the expression of LUM across different studies, including ours, could be due to differences in the Fig. 5 The protein–protein interactions network for miR-21-5p target genes retrieved from the string database (https:// string- db. org/) shows the three clusters of interconnected proteins Page 12 of 15Mahmoud et al. The Journal of Basic and Applied Zoology (2025) 86:1 types of ectopic endometrial lesions analyzed, staging of endometriosis, phases of the uterine cycle (proliferative vs. secretory), or methodological variations of detection. Although the in-silico analysis identified LUM as a putative target of miR-21-5p, our results did not reveal a negative correlation between miR-21-5p and LUM in ovarian endometrioma. Direct regulation of LUM by miR- 21-5p needs to be elucidated in a future study using an in  vitro model of endometrial cells. Our further several target genes, including LUM, of miR-21-5p are involved in multiple signalling pathways, such as MAPK, P13K-Akt, RAS, proteoglycan, TGF-beta, EGFR, and Hippo signal - ing pathways signalling pathways, known to be implicated in several relevant biological processes in endometriosis pathogenesis, such as the invasion, migration, differen - tiation, and adhesion of cells (Ahrenset al., 2020; Glaviano et  al., 2023; Huang & Chen, 2012; Iozzo & Sanderson, 2011; Molina & Adjei, 2006; Paplomata & O’regan, 2014; Wei & Liu, 2002; Zenonos & Kyprianou, 2013). The limitations that could be associated with this study are the relatively small sample size and experimental validation of the direct targeting of LUM by miR-21-5p using an in vitro model. However, some points should be considered, such as the challenges associated with diag - nosing ovarian endometriosis and its low incidence rate (Macer & Taylor, 2012; Nnoaham et al., 2011).

Overall, this study unveiled that miR-21-5p was upregulated, while its putative target LUM was downregulated, and that their dysregulated expressions may be involved in the pathogenesis of ovarian Fig. 6 A The protein–protein interactions network for LUM with other target genes identified for miR-21-5p was retrieved from the string database (https:// string- db. org/). (Accessed on 15 October 2024) B The functional pathway enrichment analysis of the proteins in the PPI network is based on the Elsevier Pathway Collection using the Enrichr database (https:// maaya nlab. cloud/ Enric hr/) (Accessed on 15 October 2024) endometriosis. We suggest that  miR-21-5p and LUM could be used as potential markers for future validation in a large cohort for ovarian endometriosis. Additional research is required to delineate the exact molecular mechanism(s) underlying the dysregulated expression of miR-21-5p and LUM in ovarian endometriosis. Abbreviations ACTB β-Actin AUC Area under the curve DAVID Database for annotation, visualization, and integrated discovery EMT Epithelial-mesenchymal transition FDR False discovery rate GO Gene ontology HESCs Endometrial stromal cells IRB Institutional review board KEGG Kyoto Encyclopedia of Genes and Genomes LUM Lumican mRNA Messenger RNA niRNA MicroRNA PDCD4 Programmed cell death 4 PPI Protein-protein interaction qPCR Quantitative real-time PCR ROC Receiver operating characteristic SEM Standard error of the mean SLRPs Short leucine-rich proteoglycans UTR Untranslated region VMP1 Vacuole membrane protein 1

This work has been performed at the Cellular and Molecular Biosciences Research Laboratory at the Department of Zoology, Faculty of Science, Cairo University. Author contributions Conceptualization: SAI and HH. Data curation: MM, AAW, SAI, and HH. Inves- tigation: MM, AAW, RSS, AB, SAI and HH. Methodology: MM, AAW, SE, MS, OA, RSS, SAI and HH. Resources: SAI. Supervision: AB, SAI, and HH. Writing – original draft: MM, AAW, RSS, SAI, and HH. Writing – review & editing: MM, AAW, MS, AS, SE, MS, OA, RSS, AB, SAI and HH. Funding Not applicable. Page 13 of 15 Mahmoud et al. The Journal of Basic and Applied Zoology (2025) 86:1 Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Declarations Ethics approval and consent to participate This study was approved by the institutional review board (IRB) at the Faculty of Medicine, Al-Azhar University (protocol number: 0000195) and the local Ethics Committee of the Faculty of Medicine at Aswan University (Protocol No. Asw.U./677/10/22). All patients who participated in the study signed a consent form to participate in this study. Consent for publication All patients who participated in the study signed a consent form to Publish in this study. Competing interests The authors declare no conflict of interest does exist. Received: 8 September 2024 Accepted: 29 November 2024

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