Extraction,Identification And Expression Of Plasma Exosomes In Patients With Atrial Fibrillation

Objective:Based on high-throughput sequencing technology to explore the microRNA(miRNA)expression in sinus rhythm(Sinus Rhythm,SR)and atrial fibrillation(Atrial fibrillation,AF)patients with plasma exosomes,looking for potential markers that may be closely related to the occurrence and development of AF,and initially explore its biological role.Methods:1.This study screened patients who went to a hospital from 2018-06 to 2019-06 as the research object,including 26 patients in the AF group and 23 patients in the SR group.Statistics of clinical data were based on the duration of AF was divided into paroxysmal AF group(PaAF,n=13)and persistent AF group(PeAF,n=13).2.Extract plasma exosomes,observe the morphology of exosomes by transmission electron microscope,particle size detection,Western blot(WB)to detect surface marker proteins.3.Three cases were randomly selected in each group,using high-throughput sequencing technology to obtain exosome miRNA differential expression profiles,searching for literature,and using bioinformatics analysis to screen out target miRNAs related to cardiovascular disease.4.For patients in the SR group(n = 20)and AF group(n = 20),Quantitative Real-time PCR(qPCR)was used to verify the target miRNA.Receiver operating characteristic curve(ROC curve)was used to evaluate the efficacy of miRNA as a marker.Results:1.Compared with the SR group,the triglyceride level in the AF group was increased,the left and right anterior and posterior diameters were enlarged,and the left ventricular ejection function was decreased.There were no significant differences in general information such as age and sex and liver and kidney function.2.Exosomes were successfully isolated from the plasma of patients with AF,and vesicles with typical exosomal structure were observed under electron microscope.Three surface marker proteins: CD9,CD63,and TSG101 were all positive.3.High-throughput sequencing results showed that compared with the SR group,there were 33 miRNAs up-regulated and 117 down-regulated in the AF group,and 4 of the up-regulated miRNAs were closely related to cardiovascular disease.4.Target gene enrichment analysis suggests that the 20 miRNA target genes that we focus on are mainly enriched in the pathways of information transmission and energy metabolism,and may exert biological functions by regulating downstream MET signaling pathway and VEGF signaling pathway.5.The qPCR results showed that the expression levels of miR-1306-5p and miR-4433b-3p were consistent with the sequencing results,and AF was significantly higher than that of SR group.ROC curve analysis suggested that miR-4433b-3p: AUC =0.885,sensitivity 80%,specificity 90%,miR-1306-5p: AUC = 0.692,sensitivity 65%,specificity 70%,miR-4433b-3p may As a marker for early diagnosis or screening for AF.Conclusions:1.Compared with the SR group,the triglyceride level in the AF group increased,the left atrium enlarged,and cardiac function deteriorated.2.There are differential miRNAs between plasma exosomes of SR group and AF group,33 miRNAs are up-regulated and 117 are down-regulated.3.Bioinformatics analysis indicates that the up-regulated miRNA mainly enriches the pathways in information transmission and energy metabolism.Related miRNA may induce the proliferation of neovascularization and exert biological functions by regulating the downstream MET signaling pathway and VEGF signaling pathway.4.Plasma exosomes miR-4433b-3p may be used as indicators for early diagnosis of AF.