A Study Of MiR-222-3p In Platelets For Auxiliary Diagnosis And Its Role In Kawasaki Vasculitis

Part Ⅰ Identification of differentially expressed microRNAs in platelet of acute Kawasaki disease.Objective:Profiling the expression of miRNAs in platelets of children with acute Kawasaki disease(KD)and other febrile diseases(OFI)by next generation sequencing technology(NGS),and screening the differentially expressed miRNAs in acute Kawasaki disease(KD),so as to further explore their role in Kawasaki vasculitis.Methods:To identify candidate miRNAs differentiating Kawasaki disease(KD)and other febrile illness(OFI),we enrolled 32 pediatric patients,including 16 children diagnosed with KD and 16 diagnosed with pneumonia,Bronchitis et al.which were grouped into OFI.In this study,we used high-throughput miRNA sequencing and found dozens of miRNAs are highly expressed in platelets.To further understand the biological significance of the upregulation of miR-222-3p in platelets of KD patients,we conducted KEGG pathway enrichment analysis of predicted miR-222-3p target genes.Three target prediction tools were chosen to identify authentic target genes of miR-222-3p,including TargetScan,miRanda and MirTarget2.A total of 165 common target genes of hsa-miR-222-3p were identified by comparing three sets of predicted target genes.DAVID was used for KEGG pathway enrichment analysis.Results:123 and 121 miRNAs were detected in platelets of OFI and KD patients,respectively,and the majority of miRNAs(78.1%)were both expressed in different disease states,including key miRNAs in innate immune responses such as miR-146a and miR-155.Among the expressed miRNAs in either KD or OFI patients’ platelets,volcano plot showed that 35 miRNAs were differentially regulated with average CPM change for more than 2 folds and p-value<0.01.12 miRNAs were upregulated in KD patients’ platelets while 23 miRNAs were downregulated.Conclusion:By comparing the miRNA expression profile of platelets of acute KD patients and other febrile patients,miR-222-3p is validated to be significantly upregulated in platelets of acute KD patients.Furthermore,KEGG pathway analysis shows that targets of miR-222-3p are enriched in immune-related signaling pathways.Our study uncovers the potential of miR-222-3p in platelets as biomarker for early diagnosis of Kawasaki disease.Part Ⅱ Establish estimation algorithm for auxiliary diagnosis of Kawasaki disease by machine learning.Objective:We try to set a estimation algorithm of Kawasaki disease through machine learning algorithm using the differentially expressed miRNAs as features,then optimize the model to improve the accuracy.Methods:We chose 9 different machining learning algorithms(Linear SVM、RBF SVM、Nearest Neighbors、Decision Tree、Random Forest、Naive Bayes、AdaBoost、LDA、QDA)to optimize the accuracy of estimation algorithm using differentially expressed 35 miRNAs as features.Then we used random forest plus linear SVM model for estimation algorithm establishment and examined the accuracy of the model by interrogating the remaining 150 samples(75 KDs and 75 OFIs).Results:We reasoned that these differentially expressed miRNAs may largely contribute to the distinguished bio-signatures of KD and OFI patients,and thus may play important roles in the early diagnosis of Kawasaki disease.A random forest classifier(RFC)model was built with a training set containing 22 samples(11 KD and 11 OFI)using differentially expressed 35 miRNAs as features.Parameters were optimized by Bayesian Optimization and used to train the model.Furthermore,we examined the generalization ability of the model by interrogating the remaining 150 samples(75 KDs and 75 OFIs).The receiver operating characteristic(ROC)curve was generated by comparing the predicted result with true sample class and the area under curve(AUC)reached 0.94,which suggested the high quality of this model in distinguishing KD from OFI samples.Conclusion:We used a random forest plus linear SVM model to build an estimation algorithm to help pediatrician for KD diagnosis and the model accuracy has reached 0.94,which suggested the high quality of this model in distinguishing KD from OFI samples.The establishment of this model laid a solid molecular biological foundation for the auxiliary diagnosis of Kawasaki disease in the early acute stage.Part Ⅲ A Study of miR-222-3p role in Kawasaki vasculitis.Objective:To predicting the target gene of miR-222-3p,we analyzed the genes,cytokines and signaling pathways related to vasculitis,and verified the regulatory relationship between related genes and cytokines expression by fluorescence quantitative PCR,so as to explore the possible mechanism of miR-222-3p in Kawasaki vasculitis.Methods:We used target gene prediction tools such as TargetScan,miRanda and MirTarget2 to predict the possible target genes of miR-222-3p,and obtained the genes,cytokines and signal pathways related to vasculitis by using DAVID analysis function and KEGG enrichment pathway.Then,we predicted the network interaction among cytokines,target genes and signaling pathways by JASPAR and cytoscape,and finally verified the expression by RT-qPCR.Results:By enriching the predicted target gene of miR-222-3p,it was found that the IL-1 family signaling pathway was in the target gene signaling pathway of miR-222-3p,and SMAD5 gene was regulated by the expression of miR-222-3p.It was predicted that the SMAD5 gene can be combined in the IL-37 gene promoter region to activate its anti-inflammatory effects.At last,the expression of miR-222-3p,SMAD5 and IL-37 in peripheral blood of KD patients,healthy children and other febrile patients was analyzed by RT-qPCR.it was found that miR-222-3p was highly expressed in KD patients,but SMAD5 and IL-37 were lower.Conclusion:By predicting the enrichment of target genes and pathways of miR-222-3p,we found that IL-1 family signaling pathway is in the target gene signaling pathway of miR-222-3p,and SMAD5 gene is regulated by miR-222-3p expression.With cytoscape and JASPAR,we predicted that the expression of the SMAD5 gene that could be combined in the IL-37 gene promoter region,and detected the expression of miR-222-3p,IL-37 and SMAD5 in blood of KD patients,healthy children,and other febrile patients by RT-qPCR.it was found that the expression trends of IL-37 and SMAD5 were the same but opposite to miR-222-3p.Therefore,it is bold to guess that the up-regulation of miR-222-3p in KD patients leads to the down-regulation of SMAD5 gene expression,and reduces the expression of IL-37.This may be a new pathogenesis of Kawasaki vasculitis,but the relationship among them needs to be well verified by cell or animal experiments.