Role And Mechanism Of UBR4 In MenSCs-exos Mediating Endometrial Repair Of IUA By Regulating YAP Ubiquitination Level

Objectives:Intrauterine adhesions（IUA）refers to a series of syndromes caused by severe endometrial basal layer injury due to traumatic uterine surgery and infection,forming fibrosis scar,causing serious deformation and partial or total blockage of uterine cavity.Intrauterine adhesions seriously damages female reproductive ability,it has become the second leading cause of secondary infertility in women.At present,there are many treatment methods for intrauterine adhesions,but the effect is not ideal.hysteroscopic electroendometriotomy（TCRA）,as a relatively common method for the treatment of IUA,has a good therapeutic effect on patients with mild and moderate intrauterine adhesions,but the postoperative recurrence rate of patients with severe intrauterine adhesions can be as high as 62.5%.It is therefore imperative to find effective and safe treatments for IUA.In recent years,Mesenchymal Stem Cells（MSCs）from various sources have been reported for the treatment of damaged endometrium.Stem cell therapy has a significant positive effect on the recovery of tissue structure and physiological function of the damaged endometrium.In recent years,the successful use of Menstrual blood-derived stromal cells（MenSCs）to improve endometrial fibrosis of intrauterine adhesions has been reported.Menstrual blood-derived stromal cells（MenSCs）have the characteristics of low immunogenicity,high proliferative ability and multidirectional differentiation potential.Previous studies of our group showed that MenSCs autologous transplantation could increase endometrial thickness and pregnancy rate in patients with severe intrauterine adhesions.Exosomes are extracellular vesicles secreted by MSCs wrapping a large amounts of protein and genetic material,which are ingested by targeted cells to exert therapeutic effects.Our group has demonstrated that MenSCs-derived exosomes（Menscs-exos）played the same therapeutic effects as MenSCs,which was both of them could restore endometrial morphology and normal physiological function in IUA rat models.Our previous mass spectrometry results showed that proteasome system molecules in MenSCs-exos were enriched in large quantities,and UBR4 was the E3 ubiquitin ligase with the highest expression.The recruitment of UBR4 is essential for the biological function of transporting and degrading proteins,but its role and mechanism in the treatment of endometrial fibrosis in IUA by MenSCs-exos remain unclear.The purpose of this study was to explore the potential molecular mechanism of UBR4 in MenSCs-exos to improve endometrial fibrosis in IUA,and to provide a theoretical basis for improving the therapeutic efficiency of MenSCs-exos in clinical application.Methods:1.The expression level of UBR4 in endometrial stromal cells（Endo SCs）of normal people and IUA patients were tested using immunofluorescence and Western-blot.UBR4 knockdown lentivirus（Sh-UBR4）was transfected into MenSCs to construct UBR4 knockdown MenSCs,and the exosomes derived from them(denoted as EXO^UBR4-)were extracted by ultracentrifugation.Exosomes derived from men SCs transfected with control lentivirus（denoted as EXO）were extracted by ultracentrifugation.The contents of CD81 and UBR4 in two exosomes were detected by Western-blot.IUA rat models established by mechanical damage were randomly divided into 3 groups(IUA group,EXO^UBR4-group and EXO group respectively),with 5 rats in each group.The rats in the IUA group were injected with 50μl PBS in each side of the uterus;the rats in EXO^UBR4-group was injected with 50μl EXO^UBR4-in each side of the uterus;the rats in EXO group were injected with 50μl EXO in each side of the uterus.After seven days of treatment,the rats in each group were weighed and killed by excessive anesthesia.The uterine tissues were collected and fixed for 48-72h before paraffin sections were made.Endometrial thickness and number of glands were observed by HE staining,endometrial fibrosis area was observed by Masson staining,and expressions of Ki67 and CD31 were detected by immunohistochemistry.2.The expression levels of fibrosis related indexes（COLⅠ,CTGF）and YAP in the endometrial of IUA rats were detected by immunohistochemistry,immunofluorescence and Western-blot.Endo SCs were extracted from IUA patients and treated with EXO^UBR4-and normal exosomes,which were denoted as IUA group,EXO^UBR4-group and EXO group respectively.Fibrosis-related indexes and YAP expression levels in Endo SCs were detected by Western-blot and immunofluorescence,and YAP ubiquitination levels were detected by Co-IP.3.MenSCs were transfected with P65 overexpression lentivirus and P65 knockdown lentivirus,and the expression level of UBR4 was detected by Western-blot.The molecular mechanism of P65 transcription regulation of UBR4 expression was clarified by double luciferase reporter gene assay and Chip assay.Results:The first part of the results showed that compared with normal people,the expression of UBR4 in Endo SCs in IUA patients was decreased.Both EXO^UBR4-and EXO expressed exosome specific surface marker CD81,and the expression level of UBR4 in EXO^UBR4-was lower than that in EXO.Animal experiments showed that,compared with IUA group,both EXO^UBR4-and EXO treatments could increase endometrial thickness and number of glands,decrease fibrosis area and enhance endometrial proliferation ability and angiogenesis ability,but the therapeutic effect of EXO^UBR4-group was significantly lower than that of EXO group.The results of the first part suggested that UBR4 was a key molecule in MenSCs-exos to improve endometrial fibrosis in IUA.The second part of animal experiments showed that both EXO^UBR4-and EXO could reduce the expression levels of fibrosis related indexes（COLⅠ,CTGF）and YAP in IUA rats endometrial,but the expression levels of COLⅠ,CTGF and YAP in EXO^UBR4-group were higher than those in EXO group.The results of cell experiments showed that the expression levels of COLⅠ,CTGF and YAP in Endo SCs of IUA patients could be decreased by both EXO^UBR4-and EXO treatment,but the expression levels of COLⅠ,CTGF and YAP in EXO^UBR4-group were higher than those in EXO group.The results of cell experiments were consistent with those of animal experiments,suggesting that knockdown of UBR4 in MenSCs-exos could weaken the ability of MenSCs-exos to reduce the endometrial fibrosis level in IUA,and UBR4 might reduce the endometrial fibrosis level in IUA by decreasing the expression level of YAP.The results of Co-IP experiment showed that UBR4 could specifically bind to YAP in Endo SCs,and the ubiquitination level of YAP in EXO^UBR4-group was lower than that in EXO group.These results indicated that UBR4 in MenSCs-exos regulated the endometrial fibrosis level of IUA by regulating the ubiquitination level of YAP.The third part of the results showed that transfection of P65 overexpression lentivirus in MenSCs could increase P65 expression level,while transfection of P65knockdown lentivirus in MenSCs could decrease P65 expression level.When P65expression level increased,UBR4 expression level also increased,and when P65expression level decreased,UBR4 expression also decreased.Ch IP experiment confirmed that P65 could bind to the UBR4 promoter region.A highly rated binding site between P65 and the UBR4 promoter region was predicted on Jaspar website,and dual luciferase gene reporting experiments confirmed that P65 could bind to the UBR4promoter region at this site.The third part suggested that P65 could regulate the expression level of UBR4 by binding to the UBR4 promoter region.Conclusions:1.This study confirmed that UBR4 was the key molecule in MenSCs-exos to improve uterine cavity morphology and reduce endometrial fibrosis level in IUA rats.2.UBR4 in MenSCs-exos inhibited endometrial fibrosis in IUA by regulating YAP ubiquitination level.3.P65 could directly bind to the UBR4 promoter region and transcriptionally regulated the expression of UBR4 in MenSCs-exos.In this study,a series of animal experiments and cell experiments were conducted to confirm that UBR4 was a key molecule in MenSCs-exos to repair IUA and inhibit endometrial fibrosis by regulating the ubiquitination level of YAP and to clarify the molecular mechanism of P65 transcriptional activation regulating UBR4 expression in MenSCs-exos.This study laid a theoretical foundation for the clinical application of MenSCs-exos in the treatment of IUA.