| Objective:1.Our objective was to find an optimized method for isolating exosomes from amniotic fluid.2.The purpose of this study was to analyze the expression of amniotic fluid(AF)-derived exosomal microRNAs and their role in TOF development.The comprehensive researches on AF-derived exosomal miRNAs allows us to understand the etiology of prenatal TOF and provide early diagnosis,allowing timely interventions.3.To investigate AF-derived exosomal non-coding RNAs and mRNAs profiling and their related networks in fetuses with VSD,we explored the competing endogenous RNA(ce RNA)regulatory network for VSD which revealed novel insights into interpreting VSD,and provided new resources for VSD diagnosis and treatment.Methods:1.Amniotic fluid was collected on the basis of prenatal multimodal ultrasound examination and fetal echocardiography.AF-derived exosomes were extracted by high-speed differential centrifugation at 110000×g and the improved centrifugation method at 20000×g respectively.Transmission electron microscopy(TEM),nanoparticle size tracking analysis(NTA)and western blot were used to characterize and assess the external appearance,the size distribution of particles consistent and the expression of marker proteins of AF-derived exosomes respectively,so as to assess the efficiency of the two methods about exosomes extraction.2.Twelve AF samples of congenital heart disease(CHD)fetuses and 12 normal AF samples were collected on the basis of prenatal multimodal ultrasound examination and fetal echocardiography.AF-derived exosomes were extracted by high-speed differential centrifugation at 110000×g and their external appearance,the size distribution of particles consistent and the expression of marker proteins were assessed by TEM,NTA and western blot respectively.3.Microarray was used to identify the differential expression profiles of AF-derived exosomal microRNAs from TOF fetuses.The candidate microRNAs were screened by bioinformatics analysis.GO and KEGG enrichment analysis were used to explore the functions of predicted target genes.Identified microRNAs were further validated by quantitative real-time polymerase chain reaction(q RT-PCR).4.miR-10a-5p and miR-200a-3p mimics were transfected into P19 cells by using Lipofectamine 3000.Western blot and q RT-PCR were applied to detect the changes of cardiac marker genes in P19 cells during differentiation into cardiomyocytes induced by dimethyl sulfoxide(DMSO).The dual luciferase reporter gene system was used to verify miR-10a-5p and its target genes.5.RNA sequencing was used to identify the differential expression profiles of AF-derived exosomal lncRNAs,microRNAs and mRNAs from VSD fetuses.The target lncRNAs of microRNAs was searched by miRanda database.The target mRNAs of microRNAs was searched by miRTar Base,Target Scan and miRDB database.The target mRNAs of lncRNAs was searched by lnc Tar D database.The coexpression analysis of lncRNAs,microRNAs and mRNAs was performed by bioinformatics method so as to construct the lncRNA-microRNA-mRNA ce RNA network.Results:1.Here,we describe a highly efficient method and optimized method for isolating exosomes from amniotic fluid.AF-derived exosomes can be obtained by high-speed differential centrifugation both at 110000×g and 20000×g.2.There was no significant difference in the biological characteristics such as particle size distribution and concentration of exosomes in AF-derived exosomes among normal fetuses,TOF fetuses and VSD fetuses.3.We identified 257 differentially expressed miRNAs from 6 AF-derived exosomes with fetal TOF using an Agilent miRNA array.KEGG pathway analysis showed that these differentially expressed microRNAs were predicted to interact with immune system and developmental biology pathway.The top 30 pathways based on gene enrichment including the Wnt and Notch signaling pathway,which has been reported to play a critical role in regulating congenital heart disease development.Then,25 out of 257 microRNAs overlapped with Notch-regulated and Wnt-regulated microRNAs simultaneously were selected as candidate microRNAs for further examination.The expression levels of 6 candidate microRNAs from microarray were verified with quantitative q RT-PCR.4.The expression of cardiac marker proteins TBX5,NKX2.5 and GATA4 were increased during cardiac differentiation of P19 cells.Overexpression of miR-10a-5p decreased the TBX5,NKX2.5 and GATA4 protein level in P19 cells.The dual-luciferase reporter assay showed that TBX5 was a direct target of miR-10a-5p.Western blot analysis showed that overexpression of miR-10a-5p inhibited the expression of TBX5 protein,which indicated that miR-10a-5p could directly target TBX5.5.We found 1090 lncRNAs,256 microRNAs and 1252 mRNAs to be differentially expressed in AF-derived exosomes from fetuses with TOF compared with healthy controls.Subsequently,the lncRNA-microRNA-mRNA competing endogenous network was constructed including 132 lncRNA–microRNA–mRNA interactions.KEGG pathway analysis showed that these differentially expressed genes in ce RNA network involved in Wnt pathway,regulating pluripotency of stem cells pathways and PI3K/Akt pathway.These core regulatory axis(lnc-PWWP2B-1:3/miR-497-5p/SUCO 、 lnc-RFXAP-2:1/miR-144-3p/KAT6 A 、lnc-GNB2L1-1-2/miR-129-5p/SIAH1)in ce RNA network were involved in the these pathways which may play a crucial role in the development of VSD.Conclusions:1.In our study,we demonstrated the presence of exosomes in the AF.We provided a straightforward and optimized method for isolating exosomes from amniotic fluid.In addition,our results indicated that there was no difference in morphology,particle size and protein markers between TOF and healthy AF-derived exosomes.2.Altered expression levels of microRNAs were found in TOF fetuses,and these differentially expressed microRNAs were predicted to interact with genes in the Notch and Wnt pathways which play a crucial role during cardiogenesis and fetal congenital heart disease development.3.We identified 257 differentially expressed microRNAs from 6 AF-derived exosome with TOF using an Agilent miRNA array.KEGG pathway analysis showed that these differentially expressed microRNAs were predicted to interact with immune system and developmental biology pathway.The top 30 pathways based on gene enrichment including the Wnt and Notch signaling pathway,which has been reported to play a critical role in regulating congenital heart disease development.Then,25 out of 257 microRNAs overlapped with Notch-regulated and Wnt-regulated microRNAs simultaneously were selected as candidate microRNAs for further examination.The expression levels of 6 candidate microRNAs from microarray were verified with qRT-PCR.4.Our study identifed the differentially expressed lncRNAs,microRNAs and mRNAs in AF-derived exosomes from VSD fetuses compared with healthy fetuses.We constructed a competitive endogenous RNA network and analysis of potential regulatory axis targets in VSD.The differential expression of ncRNAs in fetus with VSD impacted expression and translation of important genes contributing to VSD.In-depth research on AF-derived exosomal ncRNAs allows us to understand the etiology of prenatal VSD and provide a theoretical basis for clinical diagnosis of VSD. |
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