The Mechanism Of MiR-221-3p On Coronary Artery Lesions In Children With Kawasaki Disease

Kawasaki disease(KD)is an acute self-limiting febrile vasculitis of unknown etiology in children under 5 years old.It mainly affected coronary arteries,resulting in coronary artery lesions,and severe cases can lead to myocardial ischemia,infarction,and even sudden death.At present,the pathogenesis and mechanism of KD has not been completely identified,and previous studies have shown that immune dysregulation caused by pathogenic invasion is a key factor in the pathogenesis of KD,and the uncontrolled inflammatory response is the key step that activating the immune system in children with KD leading to the vascular damage.micro RNA-221 is a non-coding small RNA that is highly expressed in vascular endothelial cells and smooth muscle cells and it has been involved in pathogenesis of some diseases,such as cardiovascular diseases,inflammatory diseases,and rheumatoid arthritis.However,the role and mechanism of miR-221-3p in children with KD and its coronary artery lesions was unclear.This study aims to further elucidate the mechanism of miR-221-3p in the inflammatory response process and coronary artery injury caused by KD in the acute phase from real-world clinical case studies and in vitro and in vivo studies,in order to provide a new potential target for KD and KD-induced coronary artery lesions.Part Ⅰ The Expression of miR-221-3p in children with Kawasaki disease and its Relationship with Coronary Artery LesionsObjective: To investigate the expression level of miR-221-3p in serum of acute stage of children with Kawasaki disease(KD),to elaborate the potential relationship between miR-221-3p and the development of KD and its coronary artery lesions,and as well as the correlation between miR-221-3p and clinical laboratory variables in children with KD.Methods: Serum samples of hospitalized children with KD were collected as experimental group,and they were divided into group of KD patients with coronary artery lesions and group of 10 sex-and age-matched KD patients without CALs.According to the sensitivity of IVIG,KD patients were divided into group of sensitive to IVIG and group of resistant to IVIG.Serum samples from healthy children in the same period were selected as the healthy control group.Reverse transcription-quantitative polymerase chain reaction(RT-q PCR)was used to detect the levels of miR-221-3p,and the correlation between miR-221-3p and clinical laboratory variables in children with KD was analyzed.The Kobayashi score of IVIG response group and IVIG resistance group was calculated respectively,and the area under the operating characteristic curve(ROC)curve(AUC)of miR-221-3p was calculated in predicting IVIG resistance and coronary artery lesions in children with KD.Results:(1)A total of 29 healthy children and 55 children with KD were enrolled in this study and there was no significant difference in gender and age between this two groups.Compared with the HCs group,the serum level of miR-221-3p were significantly increased in the KD group(p < 0.05),and it was positively correlated with clinical inflammatory indicators : N%,CRP,PCT and NLR,and negatively correlated with the L%,and it was significantly association with total dosage of IVIG and administration times of IVIG in KD group(r = 0.501,p < 0.001;r = 0.558,p < 0.001,respectively),while no relationship with the onset age,gender,and weight(all p > 0.05).(2)Compared with the KD-NACLs group and IVIG-Reponse group,miR-221-3p was elevated in KD patients with CALs and IVIG resistance(p<0.05).Additionally,KD with IVIG resistance were more likely to have a longer duration of fever,a higher total dosage of IVIG,higher levels of MPV,PDW,ANC,TLB,ALT,AST,CRP,PCT,NLR,PT and ALC,PLT,and ALB than that of IVIG response group,while age,gender,weight and time point of IVIG were not different.(3)The group of IVIG resistance had a higher level of Kobayashi Score(p < 0.001).The ROC analysis showed that miR-221-3p had a better value for diagnosis IVIG resistance in children with KD than Kobayashi Score with the AUC of 0.811(95% CI,0.672–0.951),0.793(95% CI,0.618–0.968),respectively.Additionally,miR-221-3p showed an AUC value of 0.83(95% CI,0.648–1.000,p < 0.05)for the prediction of the complication of coronary artery abnormalities in the group of KD with CALs.Conclusions: miR-221-3p was significantly higher in the acute stage of Kawasaki disease than that in healthy controls in this small clinical trial,and was increased in the coronary artery lesions group and IVIG resistance group in children with KD,indicating that miR-221-3p might be involved in the pathogenesis of KD and its complication of coronary artery injury and IVIG resistance and miR-221-3p can be used as a new potential biomarker to predict coronary artery complication and IVIG resistance in children with KD.Part Ⅱ Target gene prediction and network regulation mechanism of hsa-miR-221-3p by bioinformatics analysisObjective: To explore the biological function and involved signal transduction pathways of the target gene,via the target genes predicting the network regulation mechanism of hsa-miR-221-3p,providing a potential theoretical basis for the mechanism of miR-221-3p in Kawasaki disease.Methods: Target gene prediction of hsa-miR-221-3p was performed using online databases as Target Scan,miRTar Base and miRDB,respectively.Then Wayne analysis was used to draw a venn map to get the set of intersection target genes.Cluster Profiler software was used to perform gene ontology(GO)functional annotation analysis of the predicted intersecting gene sets and database path analysis of the Kyoto Gene and Genome Encyclopedia(KEEG).The regulatory network of miR-221-3p intersection gene set is obtained by using the protein interaction network(PPI),and the key functional modules and key genes(HUB genes)in the regulatory network of miR-221-3p are found by the Cytoscape software.Results:(1)The Target Scan,miRTar Base and miRDB target gene prediction databases were used to obtain 252,485 and 616 target genes predicted by miR-221-3p respectively,and Wayne analysis of the target genes predicted by the three databases revealed a intersection target gene set with the binding sites to hsa-miR-221-3p,among which 42 common target genes accounted for 3.10% of the total predicted target genes.(2)Go functional annotation analysis showed that the hsa-miR-221-3p intersection target gene sets were mainly enriched in the microtubule related complex,endoplasmic reticulum,nuclear inclusion body,telomerase complex,phosphatidylinoinositol 3-kinase complex,cytoplasmic dynein complex and simultaneously participated in many molecular functions and biological processes,such as cell adhesion,apoptosis,DNA damage,ubiquitination of proteins,transcriptional activator activity RNA polymerase II transcription regulatory region sequence-specific binding,and so on.KEGG Pathway enrichment analysis showed that the hsa-miR-221-3p intersection target gene sets were mainly enriched in signaling pathways,such as pathways in colorectal cancer,apoptosis,tumor necrosis factor signaling pathway and so on.(3)Based on the PPI network construction of hsa-miR-221-3p intersection target gene sets,the two MOCDE and the top 10 hub genes of radiality were screened,which were FOS、CTCF、RNF4、BCL2L11、SOCS3、PIK3R1、DDIT4、DUSP16、PCDHA10、PCDHA7.According the literature search of the relationship of the top 10 HUB genes with KD,it was found that the HUB gene SOCS3 of miR-221-3p could participate in the occurrence of KD.Additionally,the KEEG enrichment analysis of miR-221-3p HUB genes showed that miR-221-3p was enriched in nonalcoholic steatohepatitis,prolactin,TNF,apoptosis,PI3K-AKT,JAK/STAT signaling pathway and so on.Conclusions: Functional enrichment analysis and signal transduction pathway enrichment analysis showed that hsa-miR-221-3p was involved in cell adhesion,apoptosis,DNA damage and protein ubiquitination.The HUB gene SOCS3 of miR-221-3p could participate in the occurrence of KD.We preliminarily predict that hsa-miR-221-3p may regulate SOCS3 mediated the inflammation of vascular damage,which provides a potential theoretical basis for the further study of the pathogenesis and treatment of KD and its coronary artery injury.Part Ⅲ Expression of miR-221-3p and its target gene SOCS3 in the coronary artery injury model of Kawasaki disease-like mouseObjective: To investigate the expression of miR-221-3p in the coronary artery injury model of Kawasaki disease-like mice and the relationship between miR-221-3p,SOCS3 and coronary artery injury.Methods: The mouse model of coronary artery injury was established by intraperitoneal injection of candida albicans water-soluble fraction(CAWS).HE staining was used to observe the inflammatory cell infiltration in the aortic and coronary artery.Aortitis and coronary angitis were used to score the vasculitis.The expression of miR-221-3p was detected by in situ hybridization.The expression of IL-6 and SOCS3 was detected by immunohistochemical method.Results:(1)The coronary artery injury model of Kawasaki disease-like mouse was successfully established by intraperitoneal injection of CAWS.HE staining showed that,compared with intraperitoneal saline group(NS group),intimal hyperplasia and morphological changes of the coronary artery was examined in CAWS induced mouse and vasculitis index of CAWS group was increased significantly(p < 0.05).(2)In situ hybridization showed that the stainning of miR-221-3p was significantly increased in CAWS group,and the expression of miR-221-3p was mainly expressed in cytoplasm and was consistent with inflammatory infiltration of blood vessels.(3)Immunohistochemistry showed that IL-6 was widely expressed in myocardial cytoplasm and less in vascular endothelial cells in NS group,and elevated expression of IL-6 was observed in CAWS induced mouse and was expressed in dilated and hyperplastic coronary arteries and overlaps with infiltrated inflammatory cells.What’s more,the expression of SOCS3 was significantly decreased in CAWS group.Conclusions: The expression of miR-221-3p and IL-6 in the CAWS model of Kawasaki disease-like mouse was significantly increased,which was elevated in the region of infiltrated inflammatory cells.Whereas the expression of SOCS3 in the downstream of miR-221-3p was decreased.It is suggested that miR-221-3p may participate in the occurrence of coronary injury in Kawasaki disease by regulating the expression of SOCS3.Part Ⅳ miR-221-3p/SOCS3 axis regulates JAK2/STAT3 Signaling Pathway mediated the inflammation of coronary artery injury in Kawasaki diseaseObjective: To identify SOCS3 as the target gene of miR-221-3p.To investigate the role of miR-221-3p in regulating SOCS3 expression and mediating the activation of JAK2/STAT3 signaling pathway in coronary artery injury in Kawasaki disease.Methods: The expression of inflammatory factors TNF-α,IL-6 and ICAM-1 were detected in human coronary artery endothelial cells(HCAECs)after affected with different concentration of TNF-α for 24 hours.The expression of miR-221-3p was detected at different time points with 10 ng/ml of TNF-α interfering HCAECs.The expression of IL-6,ICAM-1 and CCL-2 in vascular endothelial cells after TNF-α intervention with HCAECs 24 hours were detected.The mimic and inhibitor of miR-221-3p were transfected to detect the changes of vascular injury factors.The double luciferase reporter identify SOCS3 is the target gene of miR-221-3p.The expression of IL-6,ICAM-1 and CCL-2.The expression level of vasculitis factors and protein phosphorylation in JAK2/STAT3 signal pathway were detected by RT-q PCR and Western blot assay induced with AZD1480,overexpression and knockdown of SOCS3.Results:(1)Different concentration of TNF-α intervened HCAECs,the expression of TNF-α、IL-6、ICAM-1was increased and HCAECs,suggested that TNF-α intervention on HCAECs may simulate the inflammatory injury of endothelial cells.(2)The expression of miR-221-3p,IL-6,ICAM-1 and CCL2 increased after TNF α intervention in HCAEC cells.(3)The expression of IL-6,ICAM-1 and CCL-2 in HCAECs were detected by transfection of miR-221-3p mimic and inhibitor.The results showed that miR-221-3p mimic could increase the expression of inflammatory factors IL-6,ICAM-1 and CCL2 in HCAECs.In contrast,the expression of IL-6,ICAM-1 and CCL-2 decreased significantly after transfection of miR-221-3p inhibitor.(4)Double luciferase reporter gene assay showed that SOCS3 was the target gene of miR-221-3p.The regulation of miR-221-3p could induce the changes of SOCS3 m RNA level and protein level.And the upregulation of miR-221-3p may aggravate the decrease of SOCS3 protein expression after TNF-α intervention,and inhibition of miR-221-3p may improve the decrease of SOCS3 protein.(5)Transfection of SOCS3 si RNA and SOCS3 overexpression plasmid(LV-SOCS3)and JAK2/STAT3 pathway inhibitor AZD1480 into HCAECs,IL-6,ICAM-1,CCL2 m RNA level and protein phosphorylation levels of JAK2/STAT3 signaling pathway changed significantly,suggesting that SOCS3 may regulate JAK2/STAT3 signaling pathway mediated the expression of vascular endothelial inflammatory factors.Conclusion: miR-221-3p and SOCS3 may regulate the expression of IL-6,ICAM-1 and CCL2 in endothelial cell injury inflammation.The miR-221-3p/SOCS3 axis may mediates the inflammatory injury of KD coronary endothelial cells by regulating JAK2/STAT3 signaling pathways.