Exosomal miR-137-3p targets UBE3C to activate STAT3, promoting migration and differentiation into endometrial epithelial cell of human umbilical cord mesenchymal stem cells under hypoxia

Figure 1 miR-137-3p was markedly elevated in human umbilical cord mesenchymal stem cell co-cultured with hypoxia-compromised endometrial epithelial cell-derived exosomes. The culture medium from healthy endometrial epithelial cell (EEC-ex) or hypoxia-compromised EEC (EECD-ex) was gathered for the extraction of exosomes, which were subsequently incorporated into a co-culture system with human umbilical cord mesenchymal stem cell. A: A volcano plot illustrating the differentially expressed microRNAs in human umbilical cord mesenchymal stem cell subjected to EEC-ex or EECD-ex; B-D: Gene Ontology analysis (B and C) and Kyoto Encyclopedia of Genes and Genomes pathway analysis (D) of the target genes associated with differentially expressed microRNAs, as assessed by RNA sequencing; E: The quantification of miR-137-3p expression levels via reverse transcription-quantitative polymerase chain reaction. Data are represented as mean ± SD. ^aP < 0.0001 vs human umbilical cord mesenchymal stem cell group, ^bP < 0.0001 vs healthy endometrial epithelial cell + human umbilical cord mesenchymal stem cell group, n = 3. EEC: Endometrial epithelial cell; GO: Gene Ontology; HucMSC: Human umbilical cord mesenchymal stem cell; EEC-ex: Healthy endometrial epithelial cell; EECD-ex: Hypoxia-compromised endometrial epithelial cell.

Figure 2 miR-137-3p augmented the migratory and differentiation capabilities of human umbilical cord mesenchymal stem cell into endometrial epithelial cell. Human umbilical cord mesenchymal stem cells were subjected to transfection with miR-137-3p mimic or negative control. A: Transwell assays were employed to assess cellular migration. Scale bar = 100 μm, n = 3; B: Wound healing assays quantified the migratory capacity of human umbilical cord mesenchymal stem cell. Scale bar = 200 μm, n = 3; C: The protein levels of CD9, CK19, vimentin, and CD13 were elucidated using western blotting; D: Western blot analysis elucidating the protein levels of CD9, CK19, vimentin, and CD13. The results are depicted as mean ± SD. ^aP < 0.0001 vs negative control mimic group, n = 3. HucMSC: Human umbilical cord mesenchymal stem cell; NC: Negative control.

Figure 3 miR-137-3p invigorated the signal transducer and activator of transcription 3 signaling cascade. Human umbilical cord mesenchymal stem cells were transfected with miR-137-3p mimic or negative control. A: Western blot analysis of p-protein kinase B and p-mitogen-activated protein kinase protein expressions; B: Western blot evaluation of p-signal transducer and activator of transcription 3 protein levels. The findings are presented as mean ± SD. AKT: Protein kinase B; MAPK: Mitogen-activated protein kinase; STAT3: Signal transducer and activator of transcription 3; NC: Negative control.

Figure 4 miR-137-3p bolstered the migratory and differentiation propensities of human umbilical cord mesenchymal stem cell into endometrial epithelial cell, an effect mitigated by signal transducer and activator of transcription 3 inhibition. Human umbilical cord mesenchymal stem cell, transfected with miR-137-3p mimic or negative control (mimic NC), were exposed to the STAT3 inhibitor Stattic. A: Transwell assays were conducted to evaluate migratory ability. Scale bar = 100 μm, n = 3; B: Wound healing assays were utilized to assess the migratory response of human umbilical cord mesenchymal stem cell. Scale bar = 200 μm, n = 3; C: The protein levels of CD9, CK19, vimentin, and CD13. The results are illustrated as mean ± SD. ^aP < 0.0001 vs negative control mimic group, ^bP < 0.0001 vs negative control mimic group, ^cP < 0.0001 vs miR-137-3p mimic group, ^dP < 0.0001 vs negative control mimic + Stattic group, n = 3. HucMSC: Human umbilical cord mesenchymal stem cell; NC: Negative control.

Figure 5 miR-137-3p facilitated signal transducer and activator of transcription 3 activation through targeting ubiquitin protein ligase E3C. A: The potential interaction between miR-137-3p and ubiquitin protein ligase E3C (UBE3C), as predicted by miRDB; B: The comparative luciferase activities; C: Human umbilical cord mesenchymal stem cell were transfected with miR-137-3p mimic, and the mRNA expressions of UBE3C were quantified; D: Western blot analysis to ascertain the protein levels of UBE3C and signal transducer and activator of transcription 3. ^aP < 0.001 vs negative control mimic group, ^bP < 0.01 vs negative control mimic group, n = 3. Results are presented as mean ± SD. UBE3C: Ubiquitin protein ligase E3C; NC: Negative control; UTR: Untranslated region.

Figure 6 Overexpression of ubiquitin protein ligase E3C negated the stimulatory influences of miR-137-3p on human umbilical cord mesenchymal stem cell performance. Human umbilical cord mesenchymal stem cells were transfected with miR-137-3p mimic or ubiquitin protein ligase E3C overexpression construct. A: Transwell assays to examine human umbilical cord mesenchymal stem cell migration. ^aP < 0.01, negative control (NC) mimic group vs miR-137-3p mimic group, ^bP < 0.01 miR-137-3p mimic group vs miR-137-3p mimic + overexpression of ubiquitin protein ligase E3C (UBE3C-OE) group. Scale bar = 100 μm, n = 3; B: Wound healing assays to gauge cell migration. ^aP < 0.0001, NC mimic group vs miR-137-3p mimic group, ^bP < 0.0001, miR-137-3p mimic group vs miR-137-3p mimic + UBE3C-OE group. Scale bar = 200 μm, n = 3; C: Western blot analysis of p-signal transducer and activator of transcription 3 protein levels. ^aP < 0.05, NC mimic group vs miR-137-3p mimic group, ^bP < 0.05, miR-137-3p mimic group vs miR-137-3p mimic + UBE3C-OE group. n = 3. The data are expressed as mean ± SD. HucMSC: Human umbilical cord mesenchymal stem cell; NC: Negative control; UBE3C-OE: Overexpression of ubiquitin protein ligase E3C; STAT3: Signal transducer and activator of transcription 3.

Figure 7 Under hypoxic injury, endometrial epithelial cells secrete exosomes rich in miR-137-3P, which act on umbilical cord blood mesenchymal stem cells. Through the sponge adsorption effect, miR-137-3P in these exosomes adsorbs ubiquitin protein ligase E3C, thereby promoting the phosphorylation of mitogen-activated protein kinase/protein kinase B. It also promotes the signal transducer and activator of transcription 3-mediated epithelial markers CD9/CD13, which in turn promotes the differentiation of umbilical cord blood mesenchymal stem cells into endometrial epithelial cells. HucMSC: Human umbilical cord mesenchymal stem cell; UBE3C: Ubiquitin protein ligase E3C; STAT3: Signal transducer and activator of transcription 3; MAPK: Mitogen-activated protein kinase; AKT: Protein kinase B.