A Study Of The Role Of MiRNA-223-3p In Kawasaki Disease Vascular Endothelial Damage Through Regulating IL-6/IL6ST-STAT3 Signaling Pathway

Background:Kawasaki disease(KD)is an acute,self-limited systemic inflammatory vascular disease that mainly involves small and medium-sized arteries,especially coronary arteries,and a high risk factor for coronary heart disease in adults.MicroRNAs(miRNAs)are hotspots of research in recent years,regulating the functions of nearly one third of human genes and participating in important biological processes such as cell proliferation,differentiation,apoptosis and metabolism.In this study,the expression levels of miRNA-223-3p(miR-223-3p)in mononuclear cells of peripheral blood samples of children with kawasaki disease were firstly detected to verify the predicted results of the gene chip:the expression levels of miRNA-223-3p were significantly up-regulated in the tissues of kawasaki disease.Then we intend to further explore the function of miR-223-3p in the pathogenesis of kawasaki disease through in vivo and in vitro studies,and clarify the specific mechanism of miR-223-3p's influence on vascular endothelial injury by regulating interleukin-6/interleukin-6 receptor beta subunit-signal transduction and transcriptional activator 3(IL-6/IL6ST-STAT3)signaling pathway,and finally provides a reliable target for clinical diagnosis and treatment of kawasaki disease.Part I:The expression of miRNA-223-3p in peripheral blood mononuclear cells of children with kawasaki disease and its relationship with coronary artery injuryObjective:Analyze the relative expression of miRNA-223-3p(miR-223-3p),interleulin-6 receptor beta subunits messenger ribonucleic acid(IL6STmRNA)in peripheral blood mononuclear cells(PBMCs)samples,interleukin 6(IL-6),tumor necrosis factor-a(TNF-?)and intercellular adhesion molecule-1(ICAM-1)in serum samples of the different groups.Investigate the relationship between miR-223-3p and the occurrence and classification of KD.It may provides a new idea for the prevention and treatment of kawasaki disease coronary artery lesions.Methods:Kawasaki disease hospitalized children were selected as the experimental group,and were divided into the coronary artery lesions group and the non-coronary artery lesions group according to the testing results of echocardiography,and acute or subacute stage according to the course.Normal infants in children's health care out-patient department and young children who were diagnosed with virus infection after hospitalization due to fever and received related examinations were selected as the control group.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the relative expression level of miR-223-3p and IL6STmRNA in PBMCs;Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression levels of serum IL-6,TNF-?,ICAM-1 to verify the relationship between miR-223-3p and the occurrence,classification and the coronary artery lesions in kawasaki disease.Results:(1)The expression level of miR-223-3p in PBMCs in kawasaki disease coronary artery lesion group(CAL group)and non-coronary artery injury group(nCAL group)was significantly higher than that in fever control group and the normal control group in acute stage(P<0.01),and the expression level of miR-223-3p in nCAL group was higher than that in CAL group(P<0.05).However,the expression level of miR-223-3p in KD subacute stage was significantly reduced in both the CAL group and the nCAL group compared with the acute stage(P<0.01),and there was no statistical difference between the febrile control group and the normal control group(P>0.05).(2)The expression level of IL6STmRNA in PBMCs of kawasaki disease CAL group and nCAL group was significantly lower than those of fever control group and normal control group(P<0.01),and the expression level of IL6STmRNA in CAL group was lower than that in nCAL group(P<0.05).However,the expression level of IL6STmRNA in KD subacute stage was increased in both the CAL group and the nCAL group compared with that in the acute stage(P<0.05).And there was no statistical difference compared with the febrile control group and the normal control group(P>0.05).(3)The expression level of miR-223-3p in PBMCs of kawasaki disease in the acute stage was negatively correlated with the expression level of IL6STmRNA(r2=0.6803,p<0.0001).(4)The serum IL-6 expression in KD group was significantly higher than that in fever control group and normal control group(P<0.01),and it was higher in KD CAL group than that in KD nCAL group(P<0.01)by ELISA.The serum IL-6 expression in the KD group was significantly reduced in the subacute stage,and there was no statistical difference between the KD group and the normal control group(P>0.05).The serum ICAM-1 level in KD group were significantly higher than those in fever control group and normal control group(P<0.01).The level of ICAM-1 in KD CAL group were higher than that in nCAL group(P<0.05).In the meanwhile although the serum ICAM-1 levels in KD group decreased in subacute stage,they were still higher than those in febrile control group and normal control group(P<0.05).(5)The level of TNF-? in KD group was significantly higher than that in fever control group and normal control group(P<0.01)by ELISA.TNF-? level in KD CAL group was significantly higher than that in nCAL group(P<0.01).The serum TNF-? level in KD group was significantly decreased in the subacute stage compared with the acute stage(P<0.01),and there was no significant difference with the normal control group(P>0.05).Conclusion:Hsa-miR-223-3p was highly expressed in the acute stage and non-coronary artery injury groups PBMCs of children with kawasaki disease in this small sample clinical trial,suggesting that it was closely related to the occurrence and classification of kawasaki disease.The IL6STmRNA expression was significantly lower in the acute stage of kawasaki disease and the non-coronary artery injury group.Furthermore the expression level of miR-223-3p was significantly negatively correlated with the level of IL6STmRNA,suggesting that there may be some regulatory relationship between the two in kawasaki disease.We therefore speculated that miR-223-3p may be involved in its acute immune inflammatory response through the regulation of related inflammatory factors and signaling pathways,thereby reducing the expression level of ICAM-1 in serum,meanwhile regulating the expression of TNF-? and alleviating vascular endothelial injury.MiR-223-3p may become a new target for the treatment of kawasaki disease in the future.Part ?:MiRNA-223-3p can regulate vascular endothelial cell injury through IL-6/IL6ST-STAT3 signaling pathway in Kawasaki disease murine modelObjective:To verify the successful establishment of kawasaki disease murine model.Detect the relative expression of miR-223-3p,IL6STmRNA in peripheral blood mononuclear cells of mice at different time points;the relative expression of STAT3mRNA,E-selectin,ICAM-1 in mouse aorta and coronary artery samples;the protein levels of TNF-? and IL-6 in peripheral blood serum.To explore the relationship between miR-223-3p and coronary artery injury and provide reference for the application of miR-223-3p agonist in the treatment of kawasaki disease coronary artery injury.Method:First use different concentration of candida albicans water soluble substance(candida albicans water-soluble fraction,CAWS)intraperitoneal injection to construct kawasaki disease murine model,and the successful establishment of kawasaki disease murine model was verified by HE staining and transmission electron microscopy.The mice were then divided into three groups:KD group,miR-223-3p group and the sham control group according to the method of random number table.Both the KD group and the miR-223-3p group were intraperitoneally injected with CAWS 8mg(0.1ml)each one at 8 am on consecutive day(1-5)of the experiment successively,The miR-223-3p group were injected with miR-223-3p agonist(lmg/kg)via tail vein at the same time on the fifth day of the experiment,while the sham control group were intraperitoneally injected with the same volume(0.1ml)of normal saline.From 8 am of Id,3d,5d,7d and 10d after the last injection,6 mice in each group were randomly selected and peripheral blood samples were collected by the enucleation of eyeball,then the cervical dislocation was performed immediately for execution,cardiac coronary and aortic specimens were collected.RT-qPCR was used to detect the relative expressions of miR-223-3p,IL6STmRNA in PBMC,STAT3mRNA,E-selectin,ICAM-1 relative expression in mouse aorta and coronary artery samples.Serum levels of inflammatory cytokines TNF-? and IL-6 were detected by ELISA at the sametime.Results:(1)Histopathological examination and electron microscope images at each time point of endovascular immune damage caused by 8 mg(O.lml)CAWS intraperaperoneal injection in mice were consistent with the changes of vasculitis caused by human kawasaki disease.(2)the expression of miR-223-3p in PBMC of KD murine model group and the miR-223-3p group all increased gradually in the early stage of the course compared with the control group,and reached the peak on the third day,The difference was statistically significant(P<0.05).The expression level of miR-223-3p group was significantly higher than that of KD group(P<0.05).Subsequently,the expression level of miR-223-3p in PBMC of both groups gradually decreased,and recovered to the control group level from day 7(P>0.05).(3)The relative expression of IL6STmRNA in PBMC of the KD murine model group and miR-223-3p group decreased gradually compared with the control group at the early stage of the disease,the nadir reached on the third day,and the difference was statistically significant(P<0.05).The expression level of miR-223-3p group was significantly lower than that of KD group(P<0.05).After that,the expression level of IL6STmRNA in PBMC of the two groups gradually increased,and approached to the level of the control group from day 7(P>0.05).(4)The relative expression of STAT3mRNA in aortic and coronary artery specimens of the KD murine model group and the miR-223-3p group decreased gradually compared with the control group at the early stage of course,the nadir reached on day 5,and the difference was statistically significant(P<0.05).The expression level of miR-223-3p group was significantly lower than that of KD group(P<0.05),And after that,the expression level of STAT3mRNA in the serum of the two groups increased gradually,and approached to the level of the control group from day 10(P>0.05).(5)Compared with the control group,the level of E-selectin and ICAM-1 in aortic and coronary artery specimens of the KD murine model group and miR-223-3p group increased gradually,reaching its peak on the 5th day,the difference was statistically significant(P<0.05).After the seventh day,the level in the two groups all gradually decreased,and there was no significant difference between them,but it was still significantly higher than that in the control group(P<0.05).(6)TNF-? and IL-6 level in peripheral blood serum of the miR-223-3p group increased gradually compared with the control group,reached its peak on the third day.And after that,gradually increased to the level of the control group.However,The serum levels of TNF-? and IL-6 in the KD model group reached its peak on day 7.The difference was statistically significant compared with the control group(P<0.01),and then gradually decreased to the control group level on the 10th day(P>0.05).Conclusion:The model of vascular endothelial injury caused by 8mg(0.1ml)CAWS intraperitoneal injection can successfully simulate the pathological changes of human kawasaki disease,and it was determined to be the appropriate concentration of Kawasaki disease murine model finally.The KD murine model experiment show that the expression trend of miR-223-3p was contrary to that of IL6ST mRNA and STAT3 mRNA,and after the peak expression of miR-223-3p,the expression of E-selectin and ICAM-1 began to increase.It was suggested that miR-223-3p could regulate the IL6/IL6ST-STAT3 signaling pathway and thus play a protective role in vascular endothelial cell injury.Part ?:MiRNA-223-3p in the coronary artery endothelial cell injury model can regulate vascular endothelial cell injury through the IL-6/IL6ST-STAT3 signaling pathwayObjective:To verify the targeting relationship between miR-223-3p and IL6ST by luciferase reporting experiment.We use the TNF-? stimulation with different concentrations and different acting time to establish an appropriate human coronary endothelial cell(HCAECs)injury model.To verify whether the plasma of KD group could stimulate the secretion of mir-223-3p by PBMC in the acute phase.Then the HCAECs injury model was treated with miR-223-3p over expression lentivirus and knockdown virus respectively,the aim was to verify that miR-223-3p could play a protective role in regulating vascular endothelial cell injury through IL-6/IL6ST-STAT3 signaling pathway in HCAECs injury model.Methods:Luciferase reporting experiment was used,the fluorescence values of firefly luciferase and sea kidney luciferase of 3'UTR+miRNA {expression target microRNA(hsa-mir-223)vector plasmid} and 3' UTR-NC(3' UTR empty plasmid,as the target gene 3' UTR plasmid negative control)+miRNA in the two groups were compared to verify that miR-223-3p could directly bind to the 3'UTR of IL6ST.The morphological change of human coronary artery endothelial cells under TNF-? activation at different concentrations for 24 h were observed by inverted phase contrast microscope.CCK-8 assay was used to determine the relative absorbance of each group under the stimulation of different concentrations of TNF-?(representing the influence of different concentrations of TNF-? on the activity of endothelial cells).We also use it to detect the relative activity of endothelial cells at different time points under the stimulation of TNF-? at the same concentration.Finally,the appropriate TNF-? concentration and acting time to establish the coronary artery injury model were determined.Then 2ml plasma in the kawasaki disease group acute phase and 2ml plasma in the healthy control group were co-cultured with peripheral blood mononuclear cells for 48 hours respectively.After that,the relative expression of miR-223-3p in the peripheral blood mononuclear cells from the two groups were detected.The next step was to verify the effect of lentivirus infection of miRNA223 on the expression of miR-223-3p.Subsequently,HCAEC was infected with miR223-3p over expression lentivirus,negative control virus,miR223-3p knockdown virus,negative control virus,and empty cell virus respectively,combined with stimulation from 40 ng/ml TNF-? or phosphate buffer(PBS),the protein expression of IL6ST in HCAECs of different groups was detected by Western blot,the levels of IL-6,E-selectin and ICAM-1 in supernatant of each group were detected by ELISA.Finally divide the cell experiment into four groups:TNF-? group(40 ng/ml TNF-? treat for 6 hours),the miR-223-3p activator group(40 ng/ml TNF-? treat for 6 hours combined with over expression lentivirus infection),the miR-223-3p inhibitor group(40 ng/ml TNF-? treat for 6 hours combined with knockdown virus infection),NC group:PBS treated HCAECs for 6 hours;the expression level of p-STAT3/STAT3 and NF-?B p65 protein in HCAECs were detected by WES(automatic western blot quantitative analysis system).Results:(1)The luciferase report indicated that miR-223-3p could directly bind to the 3'UTR of IL6ST and inhibit the expression of IL6ST.(2)Observation by inverted phase contrast microscope:The morphology of coronary endothelial cells in the control group was normal.As the concentration of TNF-?increases,its damage to HCAECs gradually increases meanwhile.It can be manifested as obvious contraction of cells,irregular cell morphology,intercellular spaces dilatation,loose its adhesive power to the wall(TNF-? 40 ng/ml 24-hour treatment group),increased floating dead cells in supernatant,and even partial cell lysis and disappearance(TNF-? 80 ng/ml 24-hour treatment group).(3)The results of CCK-8 assay suggested that TNF-? treated with 40 ng/ml for 24 h could significantly reduce the activity of endothelial cells(CCK-8 value decreased significantly).And when the concentration of TNF-? was increased again,the value of CCK-8 changed little.(4)The results of CCK-8 assay indicated that the relative vitality of endothelial cells did not decrease significantly after treatment of 40 ng/ml TNF-? for 6 hours.Then the relative activity of endothelial cells decreased dramatically with the prolongation of treatment time.Therefore,40 ng/ml and 6 hours were selected as the appropriate concentration and acting time for further experiment of the TNF-? induced coronary artery endothelial cell injury model.(5)Compared with the healthy control group,the relative expression level of miR-223-3p in peripheral blood mononuclear cells after co-cultured with plasma in the acute phase of KD group was significantly increased(P<0.01).(6)Compared with the negative control group of the miR-223-3p over expression lentivirus,the expression level of miR-223-3p in supernatant after TNF-? stimulated HCAECs was significantly increased in the miR-223-3p over expression lentivirus group(P<0.01);Compared with the negative control group of miR-223-3p knockdown virus,the expression level of miR-223-3p in supernatant after TNF-? stimulated HCAECs was significantly reduced in the miR-223-3p knockdown virus group(P<0.05).(7)Compared with the negative control group of the miR-223-3p over expression lentivirus,the expression level of IL6ST in TNF-? stimulated HCAECs was significantly reduced in the miR-223-3p over expression lentivirus group(P<0.05);Compared with the negative control group of miR-223-3p knockdown virus,the expression level of IL6ST in HCAECs stimulated by TNF-? increased significantly in the miR-223-3p knockdown virus group(P<0.01);(8)There was no significant difference in IL-6 level in the supernatant among the three groups:TNF-? group,miR-223-3p over expression lentivirus group and knockdown virus group(P>0.05).The level of E-selectin and ICAM-1 in HCAECs of miR-223-3p over expression lentivirus group were significantly lower than those of TNF-? group(P<0.01);However,E-selectin and ICAM-1 levels in the miR-223-3p knockdown virus group were not significantly different from those in the TNF-? group(P>0.05).(9)When compared with the TNF-? group,miR-223-3p activator group can down-regulated the p-STAT3/STAT3 and NF-?B p65 protein expression level in the HCAECs(P<0.01);While the miR-223-3p inhibitor group can significantly increased the relative expression level of p-STAT3/STAT3 and NF-?B p65 proteins in HCAECs(P<0.05).Conclusion:Luciferase assay confirmed that miR-223-3p could specific bind to the 3'UTR of the target gene of IL6ST and inhibit the expression of IL6ST.The expression of miR-223-3p is closely related to the levels of IL6ST,p-STAT3/STAT3 and NF-?B p65.MiR-223-3p can regulate the expression of E-selectin and ICAM-1 in coronary artery endothelial cells by regulating the IL-6/IL6ST-STAT3 signaling pathway,alleviate the vascular endothelial injury.MiR-223-3p is an important regulatory factor for vascular endothelial cell injury in KD,and may become a potential target for the treatment of KD in the future.