ADSC Exosomes Mediate lncRNA-MIAT Alleviation of Endometrial Fibrosis by Regulating miR-150-5p

Abstract

Background: Secondary infertility remains a major complication of endometrial fibrosis in women. The use of exosomes from adipose-derived mesenchymal stem cells (ADSCs) has shown promising results for the treatment of endometrial fibrosis. However, the mechanisms of action of ADSC-exosome (ADSC-Exo) therapy remain unclear.

Materials and methods: An endometrial fibrosis model was established in mice treated with alcohol and endometrial epithelial cells (ESCs) treated with TGF-β1. ADSCs were isolated from Sprague Dawley (SD) rats, and exosomes were isolated from ADSCs using ExoQuick reagent. Exosomes were identified by transmission electron microscopy (TEM), NanoSight, and Western blot analysis. The expression level of lncRNA-MIAT was detected by qPCR analysis. Western blot analysis was carried out to determine the protein levels of fibrosis markers (TGFβR1, α-SMA, and CK19). A dual-luciferase reporter gene assay was used to verify the relationship between target genes. The endometrial tissues of the endometrial fibrosis model were stained with HE and Masson's trichrome.

Results: ADSCs and ADSC-Exos were successfully isolated, and the expression level of lncRNA-MIAT was significantly down-regulated in endometrial tissue and the TGF-β1-induced ESC injury model, whereas ADSC-Exos increased the expression of lncRNA-MIAT in the TGF-β1-induced ESC model. Functionally, ADSC-Exo treatment repressed endometrial fibrosis in vivo and in vitro by decreasing the expression of hepatic fibrosis markers (α-SMA and TGFβR1) and increasing the expression of CK19. Moreover, miR-150-5p expression was repressed by lncRNA-MIAT in the TGF-β1-induced ESC injury model. The miR-150-5p mimic promoted TGF-β1-induced ESC fibrosis.

Conclusion: ADSC-Exos mediate lncRNA-MIAT alleviation of endometrial fibrosis by regulating miR-150-5p, which suggests that lncRNA-MIAT from ADSC-Exos may be a viable treatment for endometrial fibrosis.

Keywords: ADSC; ADSC-exosomes; LncRNA-MIAT; endometrial fibrosis; miR-150-5p.

FIGURE 1

Characterization of ADSC-exosomes. (A) Western blot analysis of CD63 expression in ADSC exosomes. (B) TEM analysis of exosomes secreted by ADSCs. Scale bar = 200 nm. (C,D) The particle size of the exosomes secreted by ADSCs was measured by NanoSight LM10 and AFM5500. ADSC, adipose-derived mesenchymal stem cells; TEM, transmission electron microscopy.

FIGURE 2

LncRNA-MIAT in ADSC-Exos alleviates endometrial fibrosis. (A) Thermal map analysis of the top differentially expressed lncRNAs in endometrial samples from patients with endometrial injury. (B) qPCR analysis of the lncRNA-MIAT expression level in TGF-β1-induced ESC injury. (C) qPCR analysis of the lncRNA-MIAT expression level in TGF-β1-induced ESC injury cocultured with or without ADSC-Exos. (D) Western blot analysis of CK19, TGFβR1, and α-SMA protein expression in TGF-β1-induced ESC injury co-cultured with or without ADSC-Exos. Data are presented as the mean ± SD (n = 3). Two-tailed unpaired Student’s t-test, *P < 0.05; ***P < 0.001; ****P < 0.0001. ADSC-Exos, adipose-derived mesenchymal stem cells-exosome; qPCR, real time quantitative PCR; TGF-β1, transforming growth factor-β1; ESC, endometrial epithelial cells; CK19, cytokeratin 19; TGFβR1, homo sapiens transforming growth factor beta receptor 1; α-SMA, alpha smooth muscle Actin. SD, standard deviation.

FIGURE 3

LncRNA-MIAT in ADSC-Exos alleviates endometrial fibrosis by targeting miR-150. (A) Schematic representation of the miR-150 site in lncRNA-MIAT-3’UTR. (B) Luciferase activity was assayed in TGF-β1-induced ESC injury co-transfected with miR-150 and luciferase reporters containing lncRNA-MIAT-3’UTR. (C) Western blot analysis of CK19, TGFβR1, and α-SMA protein expression in TGF-β1-induced ESC injury co-cultured with or without ADSC-Exos in the absence or presence of miR-150 mimic. Data are presented as the mean ± SD (n = 3). Two-tailed unpaired Student’s t-test, **P < 0.01; ****P < 0.0001. ADSC-Exos, adipose-derived mesenchymal stem cells-exosome; TGF-β1, transforming growth factor-β1; ESC, endometrial epithelial cells; CK19, cytokeratin 19; TGFβR1, Homo sapiens transforming growth factor beta receptor 1; α-SMA, alpha smooth muscle actin. SD, standard deviation.

FIGURE 4

LncRNA-MIAT in ADSC-Exos alleviates mouse endometrial fibrosis by targeting miR-150. (A) The number of glands and levels of fibrosis were detected by H&E staining of the endometrial tissue from mouse endometrial fibrosis models (magnification, ×20). (B) The number of glands and levels of fibrosis were detected by Masson staining of the endometrial tissue from mouse endometrial fibrosis models (magnification, ×20). (C) The pregnancy rate was measured in each group of mouse endometrial fibrosis models. (D,E) Western blot analysis of CK19, TGFβR1 and α-SMA protein expression in the endometrial tissue of mouse endometrial fibrosis models. Data are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. H&E, hematoxylin-eosin; ADSC-Exos, adipose-derived mesenchymal stem cells-exosome; CK19, cytokeratin 19; TGFβR1, homo sapiens transforming growth factor beta receptor 1; α-SMA, alpha smooth muscle actin. SD, standard deviation.