Analysis Of Differential Expression Of CircRNAs In Serum Exosomes Of Sepsis And Preliminary Study Of Its Clinical Application

Background and objective:Sepsis is a common life-threatening organ dysfunction,with high morbidity and mortality,rapid changes in the patients' condition,and poor prognosis.Its pathogenesis is a complex dynamic process involving multiple inflammatory cells,inflammatory mediators,and cytokines Current researches find that in the early stage of sepsis,it is mainly an excessive inflammatory response,represented by a cytokine storm.In the late stage of sepsis,it gradually develops into persistent inflammation,immunosuppression and catabolism syndrome?PICS?.At present,there is no simple and clear clinical "gold standard" for clinical diagnosis.The SOFA score was recommended by the 2016 sepsis guidelines for the diagnosis of sepsis.It is mainly used to evaluate the perfusion of organs,but it cannot specifically identify the infection.The focus of sepsis treatment is early effective antibacterial treatment,fluid resuscitation and organ function support treatment,but there is still no effective molecular targeted therapy.Therefore,looking for specific diagnostic markers of sepsis and treatment targets for pathophysiological changes are of great value in delaying or reversing disease progressioncircRNA is a type of non-coding RNA produced by reverse splicing events.There are various types of circRNAs,which are widely distributed in the cytoplasm.They have the characteristics of tissue-cell specificity,high stability,and species conservation.It can be specifically enriched and stably present in exosomes,and then exert biological effects on recipient cells.Studies have found that differentially expressed circRNAs regulate the functions of signal pathway-related genes through the circRNA-microRNA-mRNA axis in many diseases.We speculate that the expression level of circRNAs changes during sepsis,which may lead to activation of multiple inflammation-related signaling pathways,and eventually cause complex inflammatory processes such as cytokine stormsIn this study,circRNA microarray technology was used to detect the expression level of circRNA in serum exosomes,R software was used to analyze differential expression,and bioinformatics was used to predict related microRNAs and mRNAs.We analyzed the pathogenesis of sepsis from the level of gene expression regulation,and initially explored the value of exosome circRNA in clinical application of sepsis combining with clinical data.Methods:Patients who were diagnosed with sepsis in the Department of Respiratory and Critical Care Medicine,Second Hospital of Jilin University from September 2018 to January 2019 were selected as the sepsis group,and healthy examinees were selected as the control group.Patients'serum samples were retained.Exosomes were extracted by ultracentrifugation.Transmission electron microscopy and western blot were used to identify exosomes.circRNA microarray technology was used to detect and analyze the serum exosome circRNAs expression profiles in 3 cases of sepsis and 3 healthy people.R software was used to analyze differential expression between the two groups.Arraystar software was used to predict microRNAs,which may be targeted and bound by the ceRNA action of circRNAs,and the circRNA-microRNA interaction was annotated in detail,in order to preliminary explore the value of differentially expressed circRNAs in clinical application of sepsis.Real-time polymerase chain reaction?qRT-PCR?was used to detect the differential expression of circRNAs between serum exosomes of sepsis group and control group in clinical samples collected later.The receiver operating characteristic curve?ROC curve?was used to evaluate the diagnostic value of hsa circRNA 104484 and hsa_circRNA₁04670 in exosomes of sepsisResults:1.Exosomes extracted from human serum were round or round-shaped "cup-shaped" under transmission electron microscopy.Western blot detection of exosomes' surface marker proteins showed CD63 and TSG101 positive2.A total of 13228 circRNAs were detected by microarray technology.With p<0.05 and Fold Change?FC?>1.5-fold selection criteria,132 differentially expressed circRNAs were selected,of which 80 circRNAs were up-regulated and 52 circRNAs were down-regulated3.In the serum exosomes of sepsis,the expression level of hsa circRNA 104484?1.829±0.718 to 1.124 ± 0.506;p=0.005?and hsa circRNA 104670[2.045?1.319-3.049?to 0.948?0.684-1.639?);p=0.003]was significantly increased,which was consistent with the results of circRNA microarray4.The ROC analysis of hsa circRNA 104484 and hsa_circRNA₁04670 was performed to evaluate the diagnostic value of exosome circRNA in sepsis.The results showed that the AUC of hsa circRNA 104484 was 0.782?95%CI:0.643-0.921;p<0.05?,the sensitivity was 0.545 and the specificity was 0.947;the AUC of hsa circRNA₁04670 was 0.775?95%Cl:0.632-0.919;p<0.05?,and the sensitivity was 0.591 and the specificity was 0.8955.We predicted microRNA targets for circRNA and looked for potential regulatory axes for circRNA.The results showed that the microRNAs targeted by hsa circRNA 104484 are hsa-miR-34b-5p,hsa-miR-508-3p,hsa-miR-378a-3p,hsa-miR-378d,hsa-miR-30c-2-3p.The microRNAs targeted by hsa circRNA 104670 are:hsa-miR-17-3p,hsa-miR-433-3p,hsa-miR-367-5p,hsa-miR-335-3p,hsa-miR-642a-5pConclusion:1.The expression of circRNAs in serum exosomes was different between patients with sepsis and healthy people2.hsa circRNA 104484 and hsa circRNA₁04670 are up-regulated in serum exosomes of sepsis,and have the potential to be used as diagnostic markers of sepsis3.In sepsis,hsa_circRNA 104484 and hsa_circRNA₁04670 may be delivered to target cells via the exosome pathway,targeting miRNA-34b-5p,miRNA-378a-3p,and miRNA-17-3p to regulate the expression levels of inflammation-related pathways,and eventually lead to pro-inflammatory,anti-inflammatory,pro-apoptotic,oxidative damage and other effects.