Mechanism Of Hypoxic UcMSCs-Derived Exosomal MiR-125b Inhibiting TP53INP1 In Endothelial Cell To Promote Wound Healing

Background:Hypoxia is a common microenvironmental feature of skin trauma.Our previous studies have shown that three-dimensional co-culture of umbilical cord-derived mesenchymal stem cells(ucMSCs)and endothelial cells(ECs)can induce intercellular communication and host integration.The purpose of this study is to clarify the interaction mechanism of ucMSCs and ECs under hypoxic conditions and their role in skin wound repair.The results of in vitro experiments illustrated that hypoxia can enhance the paracrine function mediated by exosomes derived from ucMSCs,thereby promoting the proliferation and migration of endothelial cells.In the mouse fullthickness skin injury model,exosomes can be taken up by endothelial cells to accelerate wound healing.Compared with normoxic exosomes,hypoxic exosomes have a stronger ability to promote endothelial cell proliferation and inhibit their apoptosis.We found that under hypoxic conditions,miR-125b in ucMSCs was induced to transcription and synthesis,and the expression increased.After packaged into hypoxic exosomes and transported into ECs,miR-125b binds to 3’ UTR of TP53INP1 mRNA,suppressing TP53INP1 expression,and alleviating hypoxia induced cell apoptosis.Blockage of the interaction of miR-125b and TP53INP1 attenuates the protection effect of hypoxic exosomes on hypoxia-induced cell growth arrest,migration inhibition and apoptosis.In vivo study further demonstrated that artificial agomiR-125b could accelerate wound healing.In summary,these findings reveals ucMSCs-ECs communication through exosomal-miR-125b/TP53INP1 signaling under hypoxic microenvironment and presents hypoxic exosome as a promising therapeutic strategy to enhance cutaneous repair.Objective:In this study,we investigated the protective effect of ucMSCs-derived exosomes on skin wound healing through a mouse full-thickness skin injury model,and uncoved the role and mechanism of hypoxic ucMSCs-derived exosomal miR-125b on endothelial cell function,providing a new strategy for skin wound repair of exosomes.Methods:Firstly,a mouse full-thickness skin injury model was established.The mice were randomly divided into three groups:the control group,the normoxic exosomes and hypoxic exosomes injection group,they were sacrificed 12 days after the injection.At the end of skin healing,the wound samples were collected to detect cell proliferation,migration,and apoptosis by immunohistochemistry.A skin healing curve was made at the same time.The previous results of CCK8 experiment,scratch experiment and transwell experiment showed that ucMSCs promoted the proliferation and migration of endothelial cells through paracrine.Next,ultracentrifugation was used to extract exosomes derived from ucMSCs under normoxia and hypoxia conditions.After a series of characterization and identification of exosomes,the uptake experiments,CCK8 experiments,transwell experiments,flow cytometry were used to detect the effect of ucMSCs-derived exosomes on the migration,proliferation and apoptosis of endothelial cells.Then we further explored the molecular mechanism of normoxic and hypoxic ucMSCs-derived exosomes to regulate endothelial cell functions.It has been reported that miR-23a,miR-125b,miR-21,and miR-145 in ucMSCs-derived exosomes have the highest abundance under hypoxic and normoxic conditions,and the expression level of miR-125b under hypoxic conditions significantly higher.We speculated that the abundance is closely related to the function of microRNA,so follow-up experiments mainly focus on miR-125b.The first step was to verify that the expression of miR-125b in endothelial cells was changed by qRT-PCR due to the uptake of miR-125b in normoxic and hypoxic ucMSCs-derived exosomes,rather than the increased synthesis of miR-125b in endothelial cells.Then CCK8,transwell,and flow cytometry were used to verify the effect of miR-125b in hypoxic exosomes on the proliferation,migration and apoptosis of endothelial cells.In order to explore the mechanism of miR-125b regulating endothelial cell function,we used GO,KEGG and other bioinformatics methods,combined with TargetScan,miRDB and PicTar and other databases,found the target intersection,analyzed the MicroRNA-Targets pairing and related signal pathways,selected the target of interest--TP53INP1.The directly miR-125bTP53INP1 binding was verified by luciferase assay.Then western blot and qRT-PCR were used to confirm the regulation of miR-125b on the expression of TP531NP1.Furthermore,CCK8,transwell and flow cytometry experiments verified that miR-125b in hypoxic ucMSCs-derived exosomes could target to inhibit TP53INP1 in ECs,promote cell proliferation and migration,and inhibit cell apoptosis.Finally,the mouse full-thickness skin injury model was established to determine the effect of artificial agomiR-125b on skin wound healing.At the end of skin injury healing,skin samples were collected for histochemical analysis and qRT-PCR was used to detect the expression levels of miR-125b and TP53INP1 genes of the wound.Results:1.ucMSCs-derived exosomes mediated the paracrine function of ucMSCs on cell proliferation and migration of ECs under hypoxia condition.2.Hypoxic exosomes promotes wound healing by enhancing cell proliferation and reducing cell apoptosis.3.ucMSCs-derived exosomal miR-125b,transcriptionally induced by hypoxia modulates the behaviors of ECs.4.The interaction of miR-125b and TP53INP1 is crucial for the function of hypoxic exosomes.5.Artificial miR-125b promotes cutaneous wound healing.Conclusion:Under hypoxic conditions,miR-125b of ucMSCs-derived exosomes promote the proliferation and migration of endothelial cells and inhibit their apoptosis by inhibiting the expression of the target gene TP53INP1,thereby promoting the healing of skin repair.