Extraction Of Exosomes From Serum Of Children With Kawasaki Disease And Differential Expression Of Exosomes-derived MiRNAs

Objective:To isolate the exosomes in the serum of children with acute Kawasaki Disease by ultracentrifugation,and then find out the differential expression exosomal miRNAs in serum of children with Kawasaki Disease(KD)at acute stage by using miRNAs microarray technology?Analyze miRNAs associated with Vasculitis to explore the possible pathogenesis of Kawasaki Disease?Method:Serum were collected from 12 KD children(acutephase,before using IVIG and aspirin)and 12 healthy children?Extract the exosomes from the serum and then observe the morphology of the exosomes?Total RNA was extracted and miRNA microarray chip was used to find out differentially expressed miRNAs from exosomes?Real-time quantitative PCR(RTqPCR)was used to verify the results of the chip?Targetscan,miRDB and miRWalk databases were used to predict the target genes of different miRNAs,and bioinformatics software was used to analyze the GO and KEGG genes of common target genes to find the target genes related to KD inflammatory vascular injury?Results:1.The diameter of the particles is 100,and the shape is complete,spherical and the size is uniform?It is confirmed that the particles are exosomes?2.In the acute phase of KD,33 differentially expressed exosomal miRNAs were screened by Microarray:Six significantly down-regulated miRNA(hsa-miR-125a-5p,hsa-miR-130b-5p,hsa-miR-146a-5p,hsa-miR-150-5p,hsa-miR-151a-5p,hsa-miR-151a-5p,hsa-miR-370-3p)and three significantly up-regulated miRNA(hsa-miR-122-5p,hsa-miR-21-5p,hsa-miR-4484)(Pvalue<0.05,FC>1.5)?Combined with RT-qPCR,it was found that hsamiR-122a-5p and hsa-miR-21-5p were up-regulated significantly,has-miR-125a-5p and has-miR-150-5p were down-regulanted significantly?3.By using biological analysis software,we obtain the common target genes of hsa-miR-122a-5p,hsa-miR-21-5p,hsa-miR-125a-5p and has-miR-150-5p?And then get the potential target genes associated with vasculitis:DDAH1/Olrl,TFDP2/PEG10/CBL,TNFAIP3/SMURF1 and PRKCA?Conclusion:1.The exosomes in serum can be separated and extracted effectively by ultra-speed centrifugation?2.Exosomes-derived miRNAs in the serum of KD children were significantly different in expression?3.Exosomes in Kawasaki Disease Serum may be involved in the development of Kawasaki Disease Vasculitis through their miRNAs?...