Effects of miR-214-5p and miR-21-5p in hypoxic endometrial epithelial-cell-derived exosomes on human umbilical cord mesenchymal stem cells

Figure 1 Identification of exosomes derived from endometrial epithelial cells and hypoxia-damaged endometrial epithelial cells. A: Endometrial epithelial cell exosomes were isolated from normal endometrial epithelial cells (EEC-exs) and exosomes derived from hypoxia-damaged endometrial epithelial cells were observed by transmission electron microscopy; B: Protein expression of CD63 and CD81/tetraspanin-28 in EEC-exs was measured by Western blotting; C: The particle number and average diameter of EEC-exs revealed by nanoparticle-tracking analysis. EEC: Endometrial epithelial cell.

Figure 2 Exosomes derived from hypoxia-damaged endometrial epithelial cells promote human umbilical cord mesenchymal stem cell migration and differentiation into endometrial epithelial cells. Human umbilical cord mesenchymal stem cells were cocultured with exosomes isolated from normal endometrial epithelial cells or exosomes derived from hypoxia-damaged endometrial epithelial cells. A and B: Human umbilical cord mesenchymal stem cell migration detected using wound healing assay; C: CD9, CK19, CD13, and vimentin protein levels determined by Western blotting. n = 3. ^dP < 0.0001. Data are described as the mean ± SD. HucMSC: Human umbilical cord mesenchymal stem cells; EEC-ex: Exosomes isolated from normal endometrial epithelial cells; EECD-ex: Exosomes derived from hypoxia-damaged endometrial epithelial cells.

Figure 3 Differentially expressed microRNAs in human umbilical cord mesenchymal stem cells cocultured with exosomes derived from hypoxia-damaged endometrial epithelial cells. Human umbilical cord mesenchymal stem cells were cocultured with exosomes derived from endometrial epithelial cells or exosomes derived from hypoxia-damaged endometrial epithelial cells. A: Heat map from microRNA sequencing analysis showed microRNA profiles that were differentially expressed in human umbilical cord mesenchymal stem cells treated with exosomes derived from hypoxia-damaged endometrial epithelial cells or exosomes were isolated from normal endometrial epithelial cells; B and C: Reverse transcription-quantitative polymerase chain reaction for detecting miR-214-5p and miR-21-5p. n = 3. NS: No significant difference, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. Data are described as the mean ± SD. HucMSC: Human umbilical cord mesenchymal stem cells; EEC-ex: Exosomes isolated from normal endometrial epithelial cells; EECD-ex: Exosomes derived from hypoxia-damaged endometrial epithelial cells.

Figure 4 Silencing of miR-21-5p or miR-214-5p promotes human umbilical cord mesenchymal stem cell migration and differentiation into endometrial epithelial cells. MiR-21-5p/miR-214-5p inhibitor was transfected into human umbilical cord mesenchymal stem cells. A and B: MiR-21-5p and miR-214-5p levels measured by reverse transcription-quantitative polymerase chain reaction; C and D: Cell migration determined using Transwell assay; E and F: Wound healing assay for measurement of cell migration; G and H: Protein expression of CD9, CK19, vimentin, and CD13 measured using Western blotting; I and J: Immunofluorescence staining for detecting expression of CD9, CK19, CD13, and vimentin. n = 3. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. Data are described as the mean ± SD. HucMSC: Human umbilical cord mesenchymal stem cells; NC: Normal control.

Figure 5 Signal transducer and activator of transcription 3 and protein inhibitor of activated signal transducer and activator of transcription 3 are the targets of miR-21-5p and miR-214-5p, respectively. A and B: Relative luciferase activities assessed by dual-luciferase reporter assay; C-H: MiR-21-5p, miR-214-5p, signal transducer and activator of transcription 3 (STAT3), and PIAS3 mRNA expression detected by reverse transcription-quantitative polymerase chain reaction; I: Western blot analysis of STAT3 and phospho (p)-STAT3 protein levels. n = 3. NS: No significant difference, ^aP < 0.05, ^bP < 0.01, ^dP < 0.0001. Data are described as the mean ± SD. STAT3: Signal transducer and activator of transcription 3; NC: Normal control.

Figure 6 Signal transducer and activator of transcription 3 inhibitor reverses the effects induced by miR-21-5p or miR-214-5p inhibitor on human umbilical cord mesenchymal stem cell function. Human umbilical cord mesenchymal stem cells transfected with miR-21-5p or miR-214-5p inhibitor, and treated with signal transducer and activator of transcription 3 inhibitor (Stattic). A and B: Transwell assay for detecting human umbilical cord mesenchymal stem cells migration; C and D: Wound healing assay for detection of cell migration; E and F: Protein levels of CD9, CK19, vimentin, and CD13 determined by Western blotting; G and H: Western blotting for detecting signal transducer and activator of transcription 3 and phospho (p)-signal transducer and activator of transcription 3 protein levels. n = 3. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. Data are described as the mean ± SD. HucMSC: Human umbilical cord mesenchymal stem cells; NC: Normal control; STAT3: Signal transducer and activator of transcription 3.

Figure 7 MiR-21-5p or miR-214-5p overexpression represses human umbilical cord mesenchymal stem cell migration and differentiation into endometrial epithelial cells, while signal transducer and activator of transcription 3 activator reverses these effects. Human umbilical cord mesenchymal stem cells were transfected with miR-214-5p or miR21-3p mimic, and treated with signal transducer and activator of transcription 3 activator (colivelin). A and B: Transwell assay for detecting human umbilical cord mesenchymal stem cell migration; C and D: Wound healing assay for measurement of cell migration; E and F: Western blotting for measuring CD9, CK19, vimentin, CD13, and phospho (p)-signal transducer and activator of transcription 3. n = 3. NS: No significant difference, ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. Data are described as the mean ± SD. HucMSC: Human umbilical cord mesenchymal stem cells; NC: Normal control; STAT3: Signal transducer and activator of transcription 3.