Clinical Correlation Between Circulating Exosomal MiRNA Profile And Ultrasound Diagnosis In Patients With Atherosclerosis And Development Of Exosome-mediated Drug Delivery Strategy

Background and ObjectiveCardiovascular disease is the first-most cause of death and disability worldwide.Atherosclerosis is the main cause of cardiovascular disease,accounting for 90% of the causes.Atherosclerosis is a chronic inflammatory disease with a complicated pathogenesis,involving inflammatory cell infiltration,cell proliferation and apoptosis.When atherosclerosis develops to an advanced stage,plaque rupture leads to thrombosis,leading to cardiac and cerebrovascular emergencies such as myocardial infarction or stroke.Early treatment is expected to reduce the risk of cardio-cerebral complications,but there is a lack of early diagnostic marker and treatment.Exosomes are endosome-derived nanometer-sized(30-150 nm)vesicles,which play an critical role in cell-to-cell communication.It can carry proteins,RNA,microRNAs and other substances.It directly participates in the intercellular information exchange and has the characteristics of stability and targeting.In the state of disease,the miRNAs in the exosomes secreted by cells change,which can reflect the pathological state of the source cells.In addition,exosomes are considered as good carriers of nucleic acid drugs because of their high targeting,low immunogenicity and low cytotoxicity.For miRNA/siRNA encapsulation,the miRNA/siRNA of interest could be either loaded with electroporation,or encapsulated in the donor cells transfected with the target miRNA.However,these strategies have relatively poor encapsulation efficiency.The purposes of this study include: 1)To identify serum exosome biomarkers by comparing with the ultrasound diagnosis of carotid atherosclerosis.2)To develop an intervention strategy to improve nucleic acids loading efficiency into exosomes for the prevention and treatment of atherosclerosis.MethodsPart one.Screening of diagnostic exosomal markers for atherosclerosis The subjects of this study were selected from a group of people who visited the Ultrasound Diagnosis Department of our hospital for cervical vascular ultrasound detection.Venous blood samples were obtained from patients to extract exosomes and clinical data related to atherosclerosis were collected.Samples from 12 subjects(6 of normal control and 6 of the AS patients)were analyzed by RNA-seq.Candidate miRNAs were probed by qPCR in all subjects.Correlation between miRNAs and ultrasound diagnosis was analyzed by SPSS.Part two.Construction of CD9-HuR exosomes and characterization in vitro and in vivo.Construction.Construction of CD9-HuR fusion expression vector was done by molecular cloning technology.The engineered vector or the control was transfected into exosome packaging cells.The exosomes were confirmed by gene expression,size and morphology analysis by Western blot,particle size analysis,and electron microscopy respectively.miR-155 and antimiR-155-ARE,which could be interacted with HuR were included for loading analysis.Targeted miRNAs in control or engineered exosomes were detected by real-time PCR.In vitro and in vivo experiments.DiO labeled exosomes were co-cultured with macrophage THP1,and the endocytosis of the modified exosomes by macrophages was observed by confocal imaging.DiR/DiI-labeled exosomes were injected into mice via tail vein,and the distribution of exosomes in various organs was observed by in vivo imaging system and confocal luminescence and photography were performed.Moreover,the miRNA delivery efficiency and downstream effects were examined in the heart,liver,spleen,lung,kidney and other organs,by analyzing the expression of miR-155 level and SOCS1 expression by qPCR and Western Blot.ResultsPart one.Screening of diagnostic exosomal markers for atherosclerosisIn the 127 subjects recruited,82 were AS patients and 45 were normal control.Among which,6 normal control and 6 patients were randomly selected for RNA-seq.From the RNA-seq data,173 miRNAs was significantly up-regulated while 195 miRNAs down-regulated.Moreover,in the scaled up experiments,miRNA-155 and miRNA-139 was found highly expressed in AS patients and positively correlated with the severity of atherosclerosis.Part two.Construction of CD9-HuR exosomes and characterization in vitro and in vivo.In the CD9-HuR engineered exosomes,the RNA-binding protein HuR was found localized inside of the exosomes.Moreover,the fused CD9-HuR were able to enrich the RNA with AREs(miR-155 and antimiR-155-AREs)in the exosomes.Compared with the control exosomes,The CD9-HuR exosomes loaded with targeted miRNAs significantly increased the level of target RNA in liver and spleen,and the miRNAs were functional as seen from the change of the downstream targets.Conclusions1.Our preliminary results indicate that microRNA155-5p and microRNA139-5p is probably a diagnostic marker for AS.Further study confirming whether it is a pathogenic factor,diagnostic marker or potential therapeutic target for atherosclerosis,is undergoing.2.The CD9-HuR engineered exosomes are efficient in enriching functional miRNAs,shedding light on the prevention and therapy of AS.