Exosomal MiRNA-320a Is Released From HAMSCs And Regulates SIRT4 To Prevent Reactive Oxygen Species Generation In POI

Object:Human amniotic mesenchymal stem cells(hAMSCs)were previously shown to effectively rescue ovarian function in a premature ovarian insufficiency(POI)mouse model.The therapeutic mechanism of hAMSC-derived exosomes(hAMSC-Exos)is not fully understood.This study was aimed to explore whether hAMSC-Exos had the function of restore the ovrian function of POI.Whether microRNA-320a(miR-320a)of hAMSC-Exos could rescure the ovarian function of POI and the potential mechanism.Methods:With differential centrifugation and exosome extraction kit to extract hAMSC-Exos,the characteristics of exosomes were identificated by transmission electron microscope,FACS,WB and Nanoparticle tracking analysis(NTA).In this study,the therapeutic effect of POI mouse modle and POI human granulosa cells(POIhGCs)involved in exosomes of hAMSC was explored by fluorescence-activated cell sorting(FACS),hematoxylin and eosin(H&E)staining,enzyme-linked immunosorbent assay(ELISA),western blot(WB),real-time quantitative polymerase chain reaction(RT-q PCR)and immunofluorescence(IF)experiments.Using sequencing of microRNAs with later bioinformatics analysis,RT-q PCR and luciferase report assay to detect miR-320a worked mainly in hAMSC-Exos and the target of miR320a was SIRT4.The mechanism and the ovarian function of exosomal miR-320a via Sirtuin 4(SIRT4)to inhibit reactive oxygen species(ROS)was investigated in POI mouse ovaries,oocytes and POI-hGSc using FACS,W,RT-q PCR,HE,ELISA and IF combined with knocking down miR-320a.Results:(1)In POI-hGCs and POI mouse ovaries,hAMSCs-Exos promoted cell proliferation,inhibited apoptosis,reduced the levels of SIRT4 and the related genes.(2)miR-320a played an important role in hAMSC-Exos and its target was SIRT4.(3)miR-320a of hAMSC-Exos reduced ROS level in the ovaries and oocytes of POI mouse modles and POI-hGCs and improved the function of POI ovaries,litter size and the ovarian weight.(4)miR-320a of hAMSC-Exos inhibited ROS via SIRT4,ANT2,AMPK and L-OPA1.Conclution:Our study is to illustrate the therapeutic potential of hAMSC-Exos in POI.Exosomal miR-320 plays a key role in the hAMSC-Exos-mediated effects on ovarian function via SIRT4 signaling.