Human Umbilical Cord Mesenchymal Stem Cells-derived Exosomes Promote Repair Of Endometrial Injury Via MiR-21-5p

Objective:Stem cell therapy of endometrial injury has shown some effect,offering hope for the treatment of infertility.The study found that exosomes derived from human umbilical cord mesenchymal stem cells(h UCMSCs-Exos)could replace stem cells in the treatment of endometrial injury,and relieve mifepristone-induced endometrial stromal cell(Endo SCs)injury.However,the molecular mechanism of h UCMSCs-Exos regulating endometrial repair is still unclear.Methods:1.Extract h UCMSCs-Exos to treat the endometrial injury model.Mesenchymal stem cells were cultured in exosome-free serum and cell supernatants were collected to extract h UCMSCs-Exos,which were identified by transmission electron microscopy,Particle metrix Zeta View and western blotting.Human endometrium tissue was taken to isolate endometrial stromal cells,induced by mifepristone to establish an endometrial stromal cell injury model in vitro,and after co-cultured with h UCMSCs-Exos,the morphological changes of injured cells were observed.CCK-8 and flow cytometry detected cells vability.2.The mi RNAs with potential regulatory effect of h UCMSCs-Exos on endometrial injury repair were analyzed.mi R-21-5p was screened after the sequencing data of mi RNA differential expression between endometrial tissues of IUA patients and normal people and the sequencing data of exosomal mi RNA were downloaded from GEO database.The endometrial stromal cells were divided into normal cell group(Control),mifepristone modeling group(Model),Exo-mi R-21-5p mimics group,Exo-NC group,mi R-21-5p inhibitor group,Exo-inhibitor NC group.On the basis of co-culture of h UCMSCs-Exos in the injured cells,mi R-21-5p mimics/inhibitor were transfected to overexpression/inhibition of mi R-21-5p in endometrial stromal cells.We verified the transfection efficiency and observed the effect on cell activity.3.To verify the regulation mechanism of mi RNA in h UCMSCs-Exos on the repair of damaged cells.The binding sites of mi RNA and downstream proteins were predicted by RNA22 software.Western blot and q PCR were used to confirm the regulation of mi R-21-5p on PTEN/AKT pathway in the process of h UCMSCs-Exos repairing endometrial injury.Results:1.The exosomes derived from umbilical cord mesenchymal stem cells are disc-shaped under the microscope,which is a typical lipid bilayer membrane structure.Size distribution of purified exosomes,the diameter peak was about 100 nm,the concentration was about 6.7×10~7particles/m L.The marker proteins of exosomes were detected by Western blot:transmembrane protein CD63 and cytosolic protein TSG101was positively expressed in exosomes,but negative in umbilical cord mesenchymal stem cells.2.Mifepristone induced the establishment of an in vitro endometrial injury cell model.After injury,the cell shape gradually elongated from a short spindle to a slender needle-like spindle.The results of CCK-8 showed that the cell viability also decreased significantly.The level of apoptosis in the mifepristone model group was significantly increased by Flow cytometry.After adding h UCMSCs-Exos treatment,the cell morphology was restored and gradually stretched,the cell vability increased,and the apoptosis rate decreased.3.We downloaded and analyzed the mi RNA sequencing data through the GEO database.The mi RNA levels in the endometrial tissue of IUA patients were significantly changed compared with normal people,and a total of 150 mi RNAs decreased.A total of 1174 mi RNAs were enriched in exosomes,and 86 mi RNAs were produced by the intersection of the two to make a Venn diagram.These mi RNAs may play an important role in exosomes repairing the damaged endometrium of IUA patients.Among them,mi R-21-5p was the most expressed mi RNA in exosomes.4.The expression of mi R-21-5p in cells after transfection was detected by RT-PCR,and the content of mi R-21-5p in injury endometrial stromal cells was significantly decreased compared with normal cells.The expression of mi R-21-5p in the Exo-mi R-21-5p mimics group was significantly higher than that in the negative control group(22.75±2.62 VS 2.17±0.28;P<0.05),and the expression of mi R-21-5p in the mi R-21-5p inhibitor group was significantly lower than that of the negative control group(0.79±0.11 VS 2.31±0.22;P<0.05).5.Regulate the content of mi R-21-5p in cells and observe the changes in cell vability.It was observed under inverted microscope that the state of cells in the negative control group with h UCMSCs-Exos was slightly better than that in model group.After transfection of mi R-21-5p mimics,the number of cells increased and the shape of cells tended to be short spindle of normal cells.Apoptosis was more severe after transfection with mi R-21-5p inhibitors than in the negative control group.The results of CCK-8and flow cytometry showed that the cell proliferation activity of the negative control group with exosome was higher than that of the model group,and the cell apoptosis rate decreased.After transfection of mi R-21-5p mimics,the absorbance and viability of the cells increased again,the apoptosis was greatly relieved.The repair effect of exosomes was also cancelled after transfection of mi R-21-5p inhibitor.The proliferative activity was even lower than that of mifepristone,and cell apoptosis was irreversible.6.We used RNA22 software to predict the binding sites between mi R-21-5p and protein PTEN.RT-PCR and Western blot confirmed that with the increase of mi R-21-5p,the expression of PTEN protein was suppressed and AKT phosphorylation was enhanced.When mi R-21-5p was inhibited,PTEN protein increased and cell apoptosis increased.Conclusion:Human umbilical cord mesenchymal stem cells derived exosomes can repair endometrial stromal cell injury induced by mifepristone.Through bioinformatics analysis,cellular and molecular level verification,it was confirmed that mi R-21-5p plays an important role in h UCMSCs-Exos repair endometrial injury.h UCMSCs-Exos promote cell proliferation and delay cell apoptosis by delivering mi R-21-5p to damaged endometrial cells,regulating the PTEN/AKT pathway in recipient cells.Our study provides theoretical support for the clinical application of human umbilical cord mesenchymal stem cells derived exosomes to repair endometrial injury.