Umbilical cord mesenchymal stem cell-derived exosomes reverse endometrial fibrosis by the miR-145-5p/ZEB2 axis in intrauterine adhesions.

RESEARCH QUESTION: What is the specific mechanism of umbilical cord mesenchymal stem cell-derived exosomes (UCMSC-exos) in regulating endometrial repair and regeneration? DESIGN: In this study, UCMSC-exos were harvested by differential ultracentrifugation from umbilical cord mesenchymal stem cell culture supernatant and identified with western blotting, transmission electron microscopy and nanoparticle tracking analysis. Transforming growth factor-ß1 (TGFß1) at different concentrations was used to construct the intrauterine adhesions cell model. The fibrotic markers were assessed by quantitative reverse transcription-polymerase chain reaction and western blotting. The effects of miR-145-5p over-expression on endometrial fibrosis were assessed. Dual luciferase assay was performed to verify the relationship between miR-145-5p and zinc finger E-box binding homeobox 2 (ZEB2). RESULTS: The isolated UCMSC-exos had a typical cup-shaped morphology, expressed the specific exosomal markers Alix, CD63 and TSG101, and were approximately 50-150 nm in diameter. TGFß1 at 10 ng/ml significantly promoted endometrial fibrosis, which was reversed by 20 µg/ml UCMSC-exos. Exosomal miR-145-5p ameliorated TGFß1-induced endometrial fibrosis. ZEB2 was inversely regulated by exosomal miR-145-5p as a direct target. CONCLUSIONS: UCMSC-exos might reverse endometrial stromal cell fibrosis by regulating the miR-145-5p/ZEB2 axis, representing a potential novel strategy to promote endometrial repair.

Upregulated microRNA let-7a accelerates apoptosis and inhibits proliferation in uterine junctional zone smooth muscle cells in adenomyosis under conditions of a normal activated hippo-YAP1 axis.

BACKGROUND: Let-7a is a small non-coding RNA that has been found to take part in cell proliferation and apoptosis. The hippo-YAP1 axis, known as a tumour suppressor pathway, also plays an important role in cell proliferation and apoptosis. YAP1, TAZ, and phospho-YAP1 play key roles in actions of the hippo-YAP1 axis. Adenomyosis (ADS) is a proliferative disease leading to a large uterus in patients with prolonged illness. Abnormal proliferation of smooth muscle cells (SMCs) in the uterine endometrial-myometrial junctional zone (JZ) is an important reason for developing ADS. This study aimed to explore the expression levels of let-7a and components of the hippo-YAP1 axis in SMCs in the uterine endometrial-myometrial JZ in ADS and to explore the roles of let-7a and the hippo-YAP1 axis of JZ SMC proliferation and apoptosis in ADS. METHODS: We collected JZ tissues for the primary culture of SMCs from 25 women diagnosed with ADS and 27 women without ADS. We used quantitative real-time polymerase chain reaction and western blotting to measure the mRNA and protein expression levels of let-7a, YAP1, TAZ, and phospho-YAP1 in ADS JZ SMCs. A CCK-8 assay and flow cytometry analysis of apoptosis were utilized to test the proliferation and apoptosis of JZ SMCs. The let-7a overexpression lentiviral vector GV280 was used to increase the expression level of let-7a. We added verteporfin to block the phosphorylation of components of the hippo-YAP1 axis. RESULTS: We found that the let-7a level was decreased, while the YAP1 and TAZ levels were increased in ADS JZ SMCs. Upregulated let-7a affected the expression levels of components of the hippo-YAP1 axis, accelerated apoptosis, and inhibited proliferation in JZ SMCs. Furthermore, accumulated YAP1 led to increasing proliferation of JZ SMCs after verteporfin treatment to block the phosphorylation of components of the hippo-YAP1 axis. If components of the hippo-YAP1 axis were unphosphorylated, upregulated let-7a could not inhibit the proliferation of ADS JZ SMCs. Upregulated let-7a could not activate the hippo-YAP1 axis in verteporfin treatment. CONCLUSIONS: Our findings suggest that the let-7a and hippo-YAP1 axis may act as important regulators of JZ SMCs proliferation, and upregulated let-7a may be an effective method to treat ADS.

Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells Promote Proliferation of Allogeneic Endometrial Stromal Cells.

Umbilical cord mesenchymal stem cells (UCMSCs) have been proposed as an ideal source for cell-based therapy to promote endometrial repair and regeneration. Furthermore, increasing evidence has indicated that UCMSC-derived exosomes (UCMSC-exos) act as important paracrine mediators to recapitulate the features of MSCs and may play a vital role in this process. UCMSCs and human endometrial stromal cells (ESCs) were isolated and characterized. ESCs were cocultured with UCMSCs and further assessed by flow cytometry and EdU incorporation assays. UCMSC-exos were extracted by differential ultracentrifugation and identified by western blots, transmission electron microscopy, and nanoparticle tracking analysis. The internalization of UCMSC-exos by ESCs was observed under a confocal microscope. ESCs were treated with UCMSC-exos at different concentrations and for different durations, with cell viability evaluated by CCK-8 assays. The cell cycle analysis showed that the percentage of ESCs in S phase significantly increased after coculture with UCMSCs, whereas it significantly decreased after inhibition of UCMSC-exo secretions. EdU incorporation assays also showed a similar trend. The isolated UCMSC-exos had a typical cup-shaped morphology with a monolayer membrane, expressed the specific exosomal markers Alix, CD63, and TSG101 and were approximately 60 to 200 nm in diameter. The PKH26-labeled UCMSC-exos were incorporated into ESCs. Moreover, UCMSC-exos enhanced the cell growth and viability of ESCs in a dose-dependent manner, and the effects occurred in a short period of time. UCMSC-exos promote the proliferation of ESCs in a dose-dependent manner; thus, they could be used as a potential treatment to promote endometrial repair.