Preliminary Study Of Uterine Myometrium Microenvironment-Derived Exosomes In Uterine Contraction

Background and objectiveUterine myometrial contraction plays a pivotal role in successful delivery.Abnormal contractions can lead to a variety of pathological conditions,involving fetal distress,neonatal asphyxia,postpartum hemorrhage,and even life-threatening conditions.However,the mechanisms of uterine contraction regulation have not been elucidated so far.There is no ideal strategy for the prevention and treatment of abnormal uterine contraction.To find the molecular target of uterine contraction regulation is the premise of the prevention and treatment of abnormal uterine contraction.Considered as essential mediators of intercellular signal transduction,tissue-exosomes possess reflection of tissue microenvironment and tissue specificity,participating in various biology processes and applying to some disease diagnosis and treatment.However,whether uterine myometrium,which plays a vital role in uterine contraction,can also secrete exosomes,and the role of the uterine myometrium microenvironment-derived exosomes in the regulation of uterine contraction is scarce and need to be explored.Here,we explored the proteomic and miRNA characteristics of exosomes derived from uterine myometrium,human myometrial smooth muscle cells(HMSMCs)and human umbilical vein endothelial cells(HUVECs)to investigate their role in intercellular communication and uterine contraction during labor,and to explore the molecular mechanisms in uterine contraction regulation.Method1.Characterization and cargoes of uterine myometrium microenvironment-isolated exosomes(1)Uterine myometrial tissues from human term non-labor(TNL)and term labor(TL)were collected,whose exosomes,uterine myometrial exosome in term non-labor(uTNL-Exo)and uterine myometrium exosome in term labor(uTL-Exo),were extracted and identified by nanoparticle size analysis(NTA),transmission electron microscopy(TEM)and western blotting(WB).(2)Quantitative liquid chromatography-tandem mass spectrometry(LC-MS/MS)and bioinformatics analysis of uterine myometrium exosomes were performed to screen for differentially expressed(DE)proteins;combined with proteomic data of previous human uterine myometrial tissue in the database to search for contraction-related exosomal proteins.(3)miRNA sequencing(miRNA-seq)and bioinformatics analysis of uterine myometrium exosomes were performed to find DE miRNAs and target gene prediction were also performed.(4)Exosomes isolated from maternal plasma,HMSMCs and HUVECs were identified.LC-MS/MS and miRNA-seq were conducted.(5)Combined the miRNA-seq results of myometrial exosomes,maternal plasma,HMSMCs and HUVECs to find miRNAs specifically secreted by HMSMCs and HUVECs in uterine myometrial exosomes.(6)The protein expression profiles of maternal plasma,HMSMCs and uterine myometrial tissues were combined,and the protein expression profiles of healthy male plasma were excluded to search for contraction-specific biomarkers in HMSMCs exosomal proteins,which were finally validated using plasma.(7)HMSMCs were conducted hypoxic treatment,and exosomes were extracted and the concentration was determined via NTA.(8)Cellular internalization assay was used to verify whether HMSMCs,HUVECs,decidual macrophages(DMs)and trophoblast cells(HTR8/Svneo)took up HMSMCsExo.2.Vascular endothelial cell-derived exosomes mediate uterine myometrium contraction via mir-187-3p/ATP2B4(1)TNL and TL uterine myometrial tissues were collected,and the expression of miR-187-3p was detected by Real-time quantitative Polymerase Chain Reaction(RT q PCR)and fluorescence in situ hybridization(FISH);the expression of ATP2B4 was detected by WB and immunofluorescence;(2)Double luciferase assay was used to verify whether miR-187-3p could bind to the3’UTR of ATP2B4 m RNA.(3)Lentiviral vector was used to overexpress miR-187-3p in HMSMCs,which was verified by RT-q PCR,WB and cell contraction assay.(4)The exosomes of HUVECs under normoxia and hypoxia were extracted and identified using NTA,TEM and WB.Cell internalization assay was used to verify whether HMSMCs took up HUVECs-Exo.(5)RT-q PCR was performed to detect the expression of miR-187-3p in HUVECs and HUVECs-Exo under normoxia and hypoxia.(6)HUVECs-Exo was co-cultured with HMSMCs,and the effect of HUVECs-Exo on the contractile function of HMSMCs was explored by cell gel contraction assay.Result1.Characterization and cargoes of uterine myometrium-isolated exosomes(1).Uterine myometrial tissues exosomes were successfully isolated.(2)Thirty-five proteins were significantly up-regulated and seventeen proteins were significantly down-regulated in uTL-Exo compared with uTNL-Exo.The functions of uTNL-Exo and uTL-Exo differentially expressed proteins were mainly enriched in coagulation,immunity,ion response and actin filament.Seven DE contraction-related exosomal proteins were identified.(3)Three miRNAs were significantly down-regulated and two miRNAs were significantly down-regulated in uTL-Exo compared with uTNL-Exo,whose target genes were also differentially expressed in uterine myometrial tissues.(4)The miRNA sequencing results showed that 156 miRNAs in myometrial tissue exosomes were secreted specifically by uterine smooth muscle cells and 48 miRNAs were secreted specifically by HUVECs.(5)Two proteins,GJA1 and SLC39A14,were specifically high expressed in HMSMCs-Exo and were higher in TL myometrial tissue and plasma than in the TNL group.2.Vascular endothelial cell-derived exosomes mediate uterine myometrial contraction via mir-187-3p/ATP2B4(1)Compared with TNL,miR-187-3p was significantly increased and ATP2B4 was significantly decreased in TL uterine myometrium.(2)miR-187-3p was able to bind to the 3’UTR of ATP2B4 m RNA.(3)The contraction of HMSMCs was enhanced after overexpression of miR-187-3p.(4)HUVECs secreted more exosomes during hypoxia and were taken up by HMSMCs.(5)Expression of miR-187-3p was increased in HUVECs and HUVECs-Exo under hypoxia.HUVECs-Exo under hypoxia enhanced contraction of HMSMCs.ConclusionIn this study,human uterine myometrium exosomes successfully isolated.The main cells in the microenvironment of uterine myometrium can communicate with each other via exosomes.GJA1 and SLC39A14 could be used as potential markers of uterine contractions.Vascular endothelial exosome miR-187-3p regulated uterine myometrial contraction by targeting ATP2B4.