Umbilical cord mesenchymal stem cell-derived exosomes inhibits fibrosis in human endometrial stromal cells via miR-140-3p/FOXP1/Smad axis

www.nature.com/scientificreports OPEN Umbilical cord mesenchymal stem cell‑derived exosomes inhibits fibrosis in human endometrial stromal cells via miR‑140‑3p/ FOXP1/Smad axis Mengling Song 1,5*, Lijun Ma 2, Yongzhao Zhu 3, Huimin Gao 4 & Rong Hu 1,5* Endometrial fibrosis is the histologic appearance of intrauterine adhesion (IUA). Emerging evidences demonstrated umbilical cord mesenchymal stem cell-derived exosomes (UCMSC-exo) could alleviate endometrial fibrosis. But the specific mechanism is not clear. In this study, we explored the effect of UCMSC-exo on endometrial fibrosis, and investigated the possible role of miR-140-3p/FOXP1/Smad axis in anti-fibrotic properties of UCMSC-exo. UCMSC-exo were isolated and identified. Transforming growth factor-β (TGF-β) was used to induce human endometrial stromal cell (HESC) fibrosis. Dual luciferase assay was performed to verify the relationship between miR-140-3p and FOXP1. The expressions of fibrotic markers, SIP1, and p-Smad2/p-Smad3 in HESCs stimulated with UCMSC-exo were detected by western blot. In addition, the effects of miR-140-3p mimic, miR-140-3p inhibitor and FOXP1 over-expression on endometrial fibrosis were assessed. The isolated UCMSC-exo had a typical cup-shaped morphology and could be internalized into HESCs. The expressions of fibrotic markers were significantly increased by TGF-β, which was reversed by UCMSC-exo. MiR-140-3p in UCMSC-exo ameliorated TGf-β-induced HESCs fibrosis. FOXP1 was identified as the direct target of miR-140-3p, which could inversely regulate miR-140-3p’s function on HESCs fibrosis. Furthermore, we demonstrated that miR-140-3p in UCMSC-exo regulated Smad signal pathway to exert the antifibrotic effect in HESCs. The anti-fibrotic effect of UCMSC-derived exosomes against HESC fibrosis was at least partially achieved by miR-140-3p/FOXP1/Smad axis. Keywords Exosome, Endometrial fibrosis, MicroRNA, Umbilical cord mesenchymal stem cell, Transforming growth factor-β Intrauterine adhesion (IUA) is a common endometrial disorder caused by trauma or infection. It is characterized by symptoms such as hypomenorrhea, amenorrhea, pelvic pain, infertility and recurrent abortion, etc.1. With the increase of uterine invasive procedures, the incidence of IUA has gradually increased year by year. Endometrial repair disorder and endometrial fibrosis have been regarded as the main mechanism of IUA f ormation2. Thus, finding a novel therapeutic strategy to reverse endometrial fibrosis has become an urgent problem to overcome IUA. Mesenchymal stem cells (MSCs) are a type of multipotent somatic stem cells that can be derived from various fetal and adult tissues, such as bone marrow, umbilical cord, adipose tissue, and p lacenta3. These cells possess the capability to differentiate into different cell lineages, and have great potential in promoting cell growth and tissue repair, making them highly promising in the field of regenerative m edicine4. For fibrotic diseases, MSCs 1 Department of Reproductive Medicine, General Hospital of Ningxia Medical University (The First Clinical Medical College of Ningxia Medical University), 804 Shengli Street, Xingqing Square, Yinchuan 750004, Ningxia, China. 2Department of Human Anatomy, Histology and Embryology, School of Basic Medicine, Ningxia Medical University, Yinchuan 750004, Ningxia, China. 3Institute of Medical Sciences, General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia, China. 4General Hospital of Ningxia Medical University (the First Clinical Medical College of Ningxia Medical University), Yinchuan 750004, Ningxia, China. 5These authors contributed equally: Mengling Song and Rong Hu. *email: ; Scientific Reports | (2024) 14:8321 | https://doi.org/10.1038/s41598-024-59093-5 1 Vol.:(0123456789) www.nature.com/scientificreports/ derived from different sources have been validated to promote tissue repair and inhibit fibrosis in many organs including kidney, lung, heart and s kin5–7. Recent evidences have shown that the anti-fibrotic properties of MSCs are closely related to their paracrine mechanisms8,9. In which, exosomes are the most crucial paracrine factors of MSCs. And they are nanosized particles consisting of a lipid bilayer that encapsulates biomolecules, including RNAs, DNAs, and soluble proteins10,11. Exosomes mediate intercellular communication by delivering biomolecules into target cells. Among the biomolecules carried by exosomes, microRNAs (miRNAs), small noncoding RNAs with 18–25 nucleotides in length, have attracted vast attention because of their important roles in regulating gene e xpression8. There are increasing evidences that exosomal miRNAs participated in the pathogenesis of organ fibrosis and tissue regeneration, and represent a promising treatment option for fibrotic d iseases8,12. In endometrial fibrosis, several studies have reported that MSC-derived exosomal miRNAs exhibited an anti-fibrotic effect. For instance, Tan et al.2 found that exosomes derived from bone marrow MSC (BMSCs) released miR-29a to inhibit fibrosis during endometrial repair of IUA. Xiao et al.13 demonstrated that exosomes could metastasize BMSC-derived miR-340 in endometrial stromal cells to attenuate endometrial fibrosis. Compared to BMSCs, human umbilical cord-MSCs (UCMSCs) have advantages such as obtainment by a noninvasive procedure and easiness for culture. Li et al.14 reported that UCMSC-derived exosomes(UCMSC-exo) could reverse endometrial stromal cell fibrosis by regulating the miR-145-5p/ZEB2 axis. miR-140-3p located on human chromosome 16q22.1 has been proved to regulate fibrogenesis in several models15. A study reported that up-regulating miR-140-3p could relieve renal fibrosis15. Furthermore, miR140-3p has been demonstrated to be released by UCMSCs and reduce fibrosis in rheumatoid arthritis rats16. Zhijuan Hua et al. confirmed that metformin can alleviate subretinal fibrosis through promoting miR-140-3p expression, inhibiting LIN28B and the JNK/STAT3 pathway17. miR-140-3p can also suppress fibrogenesis in TGFβ1-induced HSC-T6 cells and may be contribute to remedy for liver fi brosis18. In addition, Forkhead box (FOX) protein P1 (FOXP1) is a transcription factor with various functions including enhancing fibrosis. For instance, Xu Shao et al. demonstrated that FOXP1 enhances fibrosis during endometriosis by up-regulating Wnt signaling activity19. Moreover, FOXP1 has capability to regulate the TGF-β1-endothelin-1 pathway to inhibit pathological cardiac fibrosis and h ypertrophy20. In tissue fibrosis, TGF-β directly activates Smad signaling, triggering profibrotic gene o verexpression21. More importantly, previous studies have shown that miR-140-3p could directly interact with FOX f amily22,23. Thus, we hypothesized that the anti-fibrotic effect of UCMSC-derived exosomes was partially achieved by miR-140-3p/FOXP1/Smad axis. In this study, we identified UCMSC-exo and explored the effect of UCMSC-exo on endometrial fibrosis (...truncated)