Changes And Mechanisms Of MiRNA-9500 In Kawasaki Shock Syndrome

Part one:Changes and significance of miRNA-9500 in Kawasaki disease shock syndrome.Background:Kawasaki disease shock syndrome(KDSS)is a rapid-developing critical disease,and easy to be diagnosed falsely and missed.It is difficuLt to differentiate this disease from septic shock.miRNAs have many biological functions in the cardiovascuLar system with stable expression,which may be a key participant and reguLator in the occurrence of many diseases.Objective:The purpose of this study is to summarize the clinical characteristics of KDSS in children and find specific miRNAs that related to this disease.And explore the changes and clinical significance of related miRNAs in this disease.Methods:We collect blood samples and clinical data from different children with acute KDSS,convalescent KDSS,acute KD,and ordinary heat,respectively.Healthy children were selected as control group.The laboratory indexes of blood test were compared.The differentially expressed miRNAs in blood samples from acute KDSS and acute KD patients were screened by gene chip technology,and further verified by real-time quantitative PCR.The diagnostic value of differentially expressed miRNAs as biomarkers of KDSS was evaluated by ROC curve.ResuLts:①The laboratory indexes of WBC,PMN,TNF-α and IL-6 in KDSS group were significantly higher than those in the SS group(p<0.05).②The microarray analysis showed that 154 miRNAs were differentially expressed between the KDSS group and the KD group compared with the control group,of which 12 miRNAs were more than 10-fold differentially expressed.③There were five candidate miRNAs that were effectively and differentially expressed in both KD and KDSS blood samples by real-time quantitative PCR:miRNA-3129-3p,miRNA-4536-3p,miRNA-513a-5p,miRNA-1973,miRNA-9500;The expression of miRNA-9500 was stable and the difference was more than seven times(p<0.001).④ As a biomarker for early diagnosis of KDSS in the acute stage,the AUC(95%CI),sensitivity and specificity of miRNA-9500 were 0.82(0.702-0.938),80%and 80%,respectively.Conclusions:The inflammatory indexes of blood test were higher in the KDSS patients,and miRNA-9500 couLd be used as a marker for early identification of KD and KDSS with high sensitivity and specificity.Part two:The effects of miRNA-9500 on the function of human aortic endothelial cells.Background:Studies have shown that the vascuLar permeability is increased,and the vascuLar endothelial cell structure and function are impaired during the pathogenesis of KDSS.However,the potential mechanism is still unclear.Objective:To analyze the effects of KDSS-related miRNA-9500 on the biological function of human aortic endothelial cells(HAECs),including cell proliferation,migration and apoptosis.Methods:① miRNA-9500 expression were up/down-reguLated in HAECs by miRNA mimics/inhibitors transfection,and then observe the function changes of HAECs.Cell proliferation ability and apoptosis were detected by CCK-8 assay and flow cytometry,respectively.Cell migration ability was detected by cell scratch assay and Transwell assay.②Western blot(WB)was used to evaluate the expression levels of proteins that related to HAECs proliferation,migration and apoptosis.③ Add serum that from different group patients into HAECs medium,the survival and apoptosis status of HAECs were observed under the microscope,and the expression of miRNA-9500 was measured.ResuLts:①After miRNA-9500mimics transfection,the apoptosis of HAECs was increased,and the proliferation and migration ability were decreased.After miRNA-9500 inhibitors transfection,the apoptosis of HAECs was decreased,while the proliferation and migration ability were increased.The differences were statistically significant(p<0.001).②WB resuLts showed that the expression levels of apoptosis-related proteins(caspase 3,caspase 9)in HAECs were increased,and Bcl-2 was decreased,while the expression levels of proteins related to cell proliferation and migration(MMP2,MMP9)were decreased after miRNA-9500 mimics transfection(p<0.001).③ The apoptosis of HAECs were most obvious after adding serum from acute KDSS patients and acute KD patients under microscope,and the expression level of miRNA-9500 in KDSS was highest after adding serum from acute KDSS patients(p<0.001).Conclusion:Overexpression of miRNA-9500 may promote apoptosis and inhibit proliferation and migration of HAECs.The expression level of miRNA-9500 of HAECs was increased after adding serum from acute KDSS patients.The overexpression of miRNA-9500 may participate in the pathogenesis of KDSS by promoting the apoptosis of HAECs.Part Three:miRNA-9500 participates in the pathogenesis of Kawasaki disease shock syndrome through PI3K/Akt1 signal pathway.Background:miRNA may involve in the pathology of KDSS by acting on related target genes and pathways.Studies have shown that miRNA-9500 can bind with the 3’UTR region of Aktl and reguLate the expression of Akt1.The PI3K/Akt signaling pathway can activate eNOS,stimuLate NO release,induce vasodilation,and moduLate the vascuLar function.Objective:To analyze the mechanism of miRNA-9500 in the pathogenesis of KDSS through PI3K/AKT pathway.Methods:①Screen target genes that related to miRNA-9500 through bioinformatics technique,and investigate KDSS related signaling pathways through microarray analysis and KEGG enrichment analysis.②Investigate the relationship between miRNA-9500 and target genes by luciferase assay.③ The blank vector(control),miRNA-9500mimics,and miRNA-9500mimics+Aktl-siRNA were transfected into HAECs,and observe the proliferation,migration and apoptosis of HAECs,and the expression levels of Aktl and PI3K of three groups.④ The miRNA-9500 mimics/inhibitors were transfected into HAECs,and evaluate the mRNA and protein expression levels of Aktl and PI3K by RT-qPCR,WB and Immunofluorescence Text(IF).⑤Evaluate the mRNA and protein expression levels of Aktl and PI3K in blood samples.ResuLts:①The bioinformatic analysis showed that PI3K/AKT signal pathway is one of the most abundant pathways related to KDSS,and Aktl was the target gene one of miRNA-9500.② Luciferase assay also showed that Aktl was the target gene of miRNA-9500(p<0.001).③CCK-8 test,Scratch test,Transwell test and Flow test showed that Aktl-siRNA couLd inhibit the effects of miRNA-9500 overexpression on the HAECs proliferative ability,transfer ability and apoptosis(p<0.001).④RT-qPCR and WB showed that overexpression of miRNA-9500 couLd up-reguLate the mRNA and protein expression levels of Aktl and PI3K(p<0.001).IF analysis showed that overexpression of miRNA-9500 couLd up-reguLate the fluorescence expression levels of Aktl and PI3K(p<0.001).⑤The levels of mRNA and protein expression of Aktl and PI3K in acute KDSS group were the highest(p<0.001).Conclusion:MiRNA-9500 couLd reguLate the biological function of HAECs through PI3K/Akt signaling pathway,which may involve in the pathogenesis of KDSS.Part Four:The effectiveness of infliximab for Kawasaki disease in children:systematic review and meta-analysisBackground:Kawasaki disease(KD)is a self-limited illness that results in coronary artery aneurysms(CAAs)and threatens children’s health and lives.The therapeutic effects of single intravenous immunoglobulin gamma(IVIG)vs.infliximab(IFX)(with or without IVIG)in young children with KD remain unclear.Thus,we made a meta-analysis and systematic review,including all of the studies which have evaluated the effectiveness and safety of IFX and IVIG KD patients.Methods:The databases of the Cochrane Library,PubMed and Embase websites were searched for articles appearing from inception until December 31,2020.Clinical studies that compared IFX either as initial therapy plus IVIG or rescue therapy after IVIG(IFX group)failure compared with IVIG treatment alone(IVIG group)in treating KD patients were included.Results:The meta-analysis included nine studies characterizing 712 patients.The treatment response was significantly higher in the adjunctive IFX therapy group than in the IVIG therapy group[odds ratio(OR)2.64;95%CI:1.52-4.59;P=0.0005].Subgroup analysis,the effect of IFX therapy on treatment response is more effectiveness in the group of the high-risk KD patients than IVIG therapy(OR 6.07;95%CI:2.30-16.04;P=0.0003;random-effects model).Further analysis showed no difference in the improvement of CAAs in short-term follow-up between the two groups.However,adding IFX either as initial therapy or as additional therapy all showed an advantageous effect regarding the Δ Z score of the left anterior descending(LAD)(MD=0.29;95%CI:0.27-0.31;P<0.00001)and right coronary artery(RCA)(MD=0.24;95%CI:0.22-0.26;P<0.00001).Further,IFX exhibited significant effect on the treatment response compared with IVIG therapy in the Asian group(OR,2.84;95%CI:1.51-5.36;P=0.001;random-effects model),and the beneficial effects of IFX were given without increasing the risk of AEs.Conclusions:This meta-analysis emphasizes the importance of IFX on the treatment response in the high-risk KD patients.IFX may play a role in the Asian KD patients and prevention of progressive CAA,and does not increase the risk of AEs in KD patients.