| BackgroundViral myocarditis(VMC)is the most common type of myocarditis,mainly caused by the Coxsackievirus B(CVB).According to statistics,about 20% of sudden infant death is viral myocarditis or fatal ventricular arrhythmias caused by it.The prognosis of patients with persistent viral myocarditis is poor.Viral myocarditis is a serious threat to children's health.Chronic inflammation of myocardial cells can cause myocardial cell growth inhibition,hypertrophy,apoptosis,then induced myocardial fibrosis,dilated cardiomyopathy and heart failure.To date,we have no effective treatment for viral myocarditis,so it is helpful that we are looking for a molecular marker for the diagnosis of viral myocarditis to take effective measures to slow down or inhibit the progress of the disease.It can also be used as a molecular target for therapeutic purposes.Mi R-19b,as a member of the mi RNA family,is highly conserved in evolution and is expressed in the embryonic heart of a variety of organisms.Some studies showed that the expression level of mi R-19b was significantly up-regulated in the myocardium of patients with dilated cardiomyopathy,which suggested that mi R-19b may be involved in cardiac development in early stage.Although mi R-19b plays an important role in the development of heart,there are few studies about the relationship between mi R-19b and the occurrence and development of viral myocarditis.ObjectiveThis study was to investigate the expression of mi R-19b in the circulating blood of children with viral myocarditis and its relationship with the severity of cardiomyopathy.The expression of mi R-19b on the proliferation and apoptosis of cardiomyocytes were also investigated and the related mechanisms were studied.We hope this study will be helpful for the clinical diagnosis of viral myocarditis and the development of new targeted therapies.Method(1)In this study,50 patients with viral myocarditis from June,2014 to June,2015 were enrolled in the first Hospital of Jilin University.The level of c Tn I and CK-MB in serum were measured.The left ventricular ejection fraction(EF)and short axis shortening(FS)were measured.The expression of mi R-19b in peripheral blood samples was analyzed by fluorescence quantitative PCR.The correlations between mi R-19b expression and the above indexes were analyzed.(2)Primary SD rat cardiomyocytes were isolated and cultured,mi R-19b mimic and mi R-19b inhibitor were transfected into these cells,the expression of mi R-19b was detected by fluorescence quantitative PCR.The activity of cell proliferation was detected by CCK-8,and apoptosis was detected by flow cytometry-based analysis after CVB3 infection.The target genes of mi R-19b were predicted by bioinformatics software,TLR2 3 'UTR and TLR2' 'UTR-mut plasmid vectors containing luciferase genes were constructed.Then these vectors were transfected with mi R-19b mimic into neonatal rat cardiomyocytes,and the luciferase activity was evaluated in these cells.On the other hand,the expression of TLR2 m RNA and protein in cells after CVB3 infection.(3)The mi R-19b overexpressing viral myocarditis model was constructed to detect the expression of mi R-19b in the myocardium of the model rats.The pathological changes of myocardium were observed by HE staining.TUNEL staining was used to detect the apoptosis of cardiomyocytes.The expression of TLR2 protein was detected by western blot.Results(1)In the severe VMC group,the expression of mi R-19b in the recovery phase was significantly higher than that in the acute phase;In the mild VMC group,the expression of mi R-19b in the recovery phase was significantly higher than the acute phase.The level of c Tn I in plasma of children with viral myocarditis was significantly higher than that in control group.In the severe VMC group,the level of c Tn I in convalescence was significantly lower than that in acute phase;in severe VMC group,the level of c Tn I was significantly lower than that in acute phase.The level of CK-MB in the plasma of children with viral myocarditis was significantly higher than that in the control group.In the severeVMC group,the level of CK-MB was significantly lower in recovery phase than in acute phase.In the mild VMC group,the level of CK-MB was significantly lower in recovery phase than in acute phase.EF was significantly lower in severe children than in control group.There was no significant difference between the severe VMC group and the control group in recovery phase.There was no significant difference between the mild VMC group and the control group in actue phase,and in recovery phase.The expression of mi R-19b was negatively correlated with serum c Tn I in acute phase of severe VMC.There was no significant correlation between the level of mi R-19b and serum c Tn I in recovery phase of severe VMC.There was no significant correlation between the level of mi R-19b and serum c Tn I in acute phase of mild VMC.There was no significant correlation between the level of mi R-19b and serum c Tn I in recovery phase of mild VMC.There was no significant correlation between the levels of mi R-19b and serum c Tn I in the control group.The expression of mi R-19b was significantly negatively correlated with the serum level of CK-MB in severe VMC.There was no significant correlation between the level of mi R-19b and the serum levels of CK-MB in recovery phase in severe VMC.There was no significant correlation between the level of mi R-19b and the serum levels of CK-MB in acute phase in mild VMC group.There was no significant correlation between the level of mi R-19b and the serum levels of CK-MB in recovery phase in mild VMC group.There was no significant correlation between the level of mi R-19b and the level of serum CK-MB in the control group.There was a significant positive correlation between mi R-19b levels and EF in acute phase of in severe VMC group,and no significant correlation between mi R-19b levels and EF in recovery phase.There was no significant correlation between the levels of mi R-19b and EF in acute phase in mild VMC group,and there was no significant correlation between mi R-19b level and EF in recovery phase in mild VMC group.There was no significant correlation between the level of mi R-19b and serum EF in the control group.There was a significant positive correlation between the levels of mi R-19b and FS in acute phase in severe VMC,but there was no significant correlation between the levels of mi R-19b and FS in recovery phase in severe VMC.There was no significant correlation between the levels of mi R-19b and FS in acute phase in mild VMC.There was no significant correlation betweenthe levels of mi R-19b and FS in recovery phase in mild VMC group.There was no significant correlation between the level of mi R-19b and the level of serum FS in control group.(2)The results show that the expression of mi R-19b in cardiomyocytes with mi R-19b mimic transfection was higher than the expression in control group;while the expression of mi R-19b in cardiomyocytes with mi R-19b inhibitor transfection was significantly lower than the control group,the difference was significant.The results showed that the cell viability of mi R-19b mimic group was higher than that of the control group at48 h,72h and 96 h,the difference was significant;while the cell viability of mi R-19b inhibitor group was lower than that of the control group at 48 h,72h and 96 h,the difference was significant.It suggests that mi R-19b has a protective effect on the proliferation of cardiomyocytes after CVB3 infection.Compared with the control group,the apoptosis rate of mi R-19b mimic group decreased obviously,and the difference was significant;while the apoptosis rate of mi R-19b inhibitor group was significantly higher than that of control group,the difference was significant.It suggests that mi R-19b can inhibit cardiomyocyte apoptosis induced by CVB3 infection.TLR2 is the target gene of mi R-19b.TLR2 3'UTR and TLR2 3'UTR-mut luciferase reporter plasmids were constructed,then were co-transfected with mi R-19b mimic into rat cardiomyocytes,.On the other hand,the blank plasmid vector was transfected into rat cardiomyocytes as NC group.Then luciferase activity was detected in each group after 48 h.In rat cardiomyocytes transfected with mi R-19b and TLR2 3 'UTR reporter plasmid,luciferase activity was sighificantly lower than that in cells of NC group.However,in rat cardiomyocytes transfected with mi R-19b and TLR2 3 'UTR-mut reporter plasmid,luciferase activity was not change significantly.These results suggest that mi R-19b regulates the expression of TLR2 gene.by targeting its 3 'UTR.The expression of TLR2 m RNA in mi R-19b mimic group was significantly lower than that in control group;The expression of TLR2 m RNA in mi R-19b inhibitor group was significantly higher than that in control group,and the difference was statistically significant.The results showed that the expression of TLR protein in mi R-19b mimic group was significantly lower than the expression in control group,the difference was statistically significant;The expression ofTLR protein in mi R-19b inhibitor group was significantly higher than that in control group,the difference was statistically significant.(3)The expression level of mi R-19b was significantly higher in the myocardial tissue of transgenic rats than in the control group.Normal SD rats infected with CVB3 after a large number of myocardial cells disintegrated,the nucleus and cell contours disappeared in the myocardial cell gap can be observed in a large number of inflammatory cell infiltration,and the infiltration of myocardial fiber disorder,myocardial tissue is destroyed.The number of myocardium disintegration was significantly decreased after CVB3 infection in myocarditis transgenic rats,and the nucleus and cell contours were clear.A small amount of inflammatory cell infiltration was observed in the gap between myocardial cells.The apoptotic rate of myocardium was significantly lower in CVB3-infected myocarditis than in normal SD rats.The expression of TLR2 protein in myocardium of transgenic rats infected with CVB3 was significantly lower than that of normal SD rats.ConclusionThe expression of mi R-19b in cardiomyocytes was significantly decreased when CVB3 infection caused viral myocarditis.Mi R-19b can inhibit the occurrence and development of viral myocarditis.Mi R-19b can directly inhibit TLR2 expression,which may be one of the mechanisms of mi R-19b protecting myocardial cells. |
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