| Viral myocarditis(VMC)is one of the most common cardiovascular disease which is a serious threat to patients' life.There are 4% to 20% patients with VMC died of sudden cardiac death each year,especially in the young.Many kinds of virus can cause viral myocarditis,such as intestinal virus,and the coxsackie B group virus type 3(Coxsackievirus B,CVB3)is the most common one in human.A large part of patients could completely recover from viral myocarditis.There are about 10%-15% of patients have residual myocardial injury and developed dilated cardiomyopathy(DCM).The pathological mechanism is not very clear.Based on the previous reports,the process consists of the following stages: in the first stage,the virus directly damage to cardiomyocyte;in the second stage,secondary cell or humoral immune response to the injury of myocardial cells;in the third stage,the damage of myocardial remodeling and DCM phase.In this continuous pathophysiology process,virus replication,myocardial infiltration of inflammatory cells and myocardial fibrosis is three important mechanisms of CVB3-induced VMC process.In the VMC infected by CVB3,miRNAs were found to play a vital role in the pathogenesis of myocarditis,and the activity of NF-?B signal pathway was changed during the pathogenesis of myocarditis.Tumor necrosis factor alpha-inducible protein 3(TNFAIP3)can regulate the activity of NF-?B signal pathway.We used bioinformatics tools TargetScan found that the 3 'UTR region of TNFAIP3 may be the direct target of mi R-19 a/b.Many studies has proved that all small RNAs are associated with Argonaute(AGO)family proteins to form RNA-induced silencing complexes(RISC)to play their role,then what the effect of Argonaute protein is in VMC,and What the relationship is between these molecules in the VMC,which is very important for studying the mechanism of VMC.Based on the research background above,we systematically investigated the mechanism of viral myocarditis in three aspects such as animal,cell and molecular levels.Part ? Effect of Coxsackievirus B Virus Type 3 Virus on Argonaute Protein Expression in Myocardium[Obeject] To investigate the mRNA and proteins expression of Argonaute(AGO)1-4 protein after Coxsackie group B virus(CVB3)infection in myocardial tissue.[Methods] 60 male inbred Balb/c mice,6 weeks old(18-22g),divided into Mock group(without any treatment),the control group(not infected with the virus but injection of PBS),CVB3 infection group(CVB3 induced VMC).At the day0,4,8 time points,we harvested the heart tissue and detected the AGO1-4 expression of RNA with polymerase chain reaction(RT-PCR)and protein level with Western Blot separately.[Results] The results of RT-PCR showed that in CVB3 group the m RNA expression of AGO1(8.89 ± 0.96 vs 1.35 ± 0.23,P <0.001)and AGO3(4.98 ± 0.36 VS 1.01 ± 0.16,P<0.001)was up-regulated and the expression of AGO2(1.12 ± 0.85 vs 0.73 ± 0.50,P = 0.102)and AGO4(0.90 ± 0.63 VS 0.82 ± 0.56,P = 0.706)was not different compared to control group after 4days of CVB3 infection.The mRNA expression of AGO1(7.01 ± 1.02 vs 1.31 ± 0.51,P <0.001)and AGO3(3.65 ± 0.29 vs0.85 ± 0.11,P<0.001)was up-regulated and AGO2(0.75 ± 0.35 vs 0.93 ± 0.55,P =0.289)and AGO4(1.15 ± 0.57 VS 1.11 ± 0.45,P = 0.821)were not different after 8day of CVB3 infection.Western Blot result showed that the protein expression of AGO1(4.52 ± 0.35 vs 0.89 ± 0.21,P<0.001)and AGO3(3.99 ± 0.33 VS 1.13 ± 0.26,P<0.001)were up-regulated compared with control group,after 4 days of CVB3 infection,while AGO2(1.16(0.97 ± 0.45 vs 0.85 ± 0.45,P = 0.155)and AGO4(1.02± 0.58 VS 0.92 ± 0.49,P = 0.591)was no significant change.The protein expression of AGO1-4 after 8 days of CVB3 infection has the similar result compared to the results after 4 days of CVB3 infection.[Conclusion] The results of animal experiments showed that the mRNA and protein expression of AGO1 and AGO3 was increased in the myocardium of mice infected with CVB3 virus,while there was no change in the mRNA and protein expression of AGO2 and AGO4.We infer that AGO1 and AGO3 are likely to play an important role in the pathogenesis of mice VMC induced by CVB3,while AGO2 and AGO4 may not participate in this process.The up-regulation of AGO1 and AGO3 protein expression in mice myocardium may be the result of myocardial injury caused by CVB3 virus infection and may also be the reason for further myocardial injury after CVB3 infection.Part ? Expression of Argonaute protein and TNFAIP3 protein in cardiomyocytes infected by Coxsackie B virus type 3 virus[Object] To investigate the expression of Argonaute protein and TNFAIP3 protein in neonatal mice cardiomyocytes infected with Coxsackie B virus type 3 virus(CVB3).[Methods] The male inbred Balb/ c neonatal mice cardiomyocytes infected by CVB3 after 3 days of isolated and cultured.The group without CVB3 infection was set as the control group.The mRNA and protein of Argonaute protein and TNFAIP3 in cardiomyocytes were extracted at 0 hour and 8 hours after CVB3 infection.Polymerase chain reaction(PCR)was used to detect mRNA expression levels of AGO1,AGO2,AGO3 and AGO4.Protein expression levels of AGO1,AGO2,AGO3,AGO4 and TNFAIP3 were detected by Western blotting.[Results] The results showed that there was no significantdifferencein the mRNA expression of AGO1(1.11 ± 0.25 VS 0.98 ± 0.21,P = 0.1043),AGO2(1.02 ± 0.29 vs1.01 ± 0.24,P = 0.1043),AGO3(1.01 ± 0.15 vs 0.99 ± 0.13,P = 0.6543),AGO4(0.13± 0.15 vs 0.97 ± 0.17,P = 0.1067)between the control group and CVB3 virus group after 0 hours of CVB3 infection.After 8 hours of CVB3 infection,the expression of AGO1(2.85 ± 0.36 vs 0.98 ± 0.13,P<0.01)and AGO3(2.35 ± 0.29 vs 1.03 ± 0.11,P<0.01)were significantly up-regulated,while the level of AGO2(1.02 ± 0.15 vs 1.01± 0.13,P = 0.8916)and AGO4(0.96 ± 0.14 VS 0.99 ± 0.16,P = 0.5669)was no significant difference.The results of Western Blot showed that the protein level of AGO1(0.90 ± 0.22 vs 0.91 ± 0.21,P = 1.000),AGO2(0.96 ± 0.25 vs 1.01 ± 0.25,P =0.5622),AGO3(1.15 ± 0.26 vs 0.97 ± 0.26,P = 0.0508),AGO4(1.01 ± 0.28 vs 0.99± 0.28,P = 0.5108)did not havesignificant difference between control group and CVB3 virus group after 0 hours of CVB3 infection.After 8 hours of CVB3 infection,AGO1(3.32 ± 0.36 vs 0.98 ± 0.13,P<0.01)and AGO3(3.19 ± 0.33 vs 1.15 ± 0.23,P<0.01)were significantly up-regulated,while AGO2(1.06 ± 0.30 vs 1.1 ± 0.35,P =0.7247)and AGO4(0.99 ± 0.21 VS 1.12 ± 0.22,P = 0.0871)protein was no significant difference.To investigate whether the expression of TNFAIP3 protein was changed after CVB3 infection,wedetectedTNFAIP3 protein expression levelafter 0and 8 hours of CVB3 infection.Results showed that the expression of TNFAIP3 protein was almost unchanged(1.32 ± 0.25 vs 1.2 ± 0.32,P = 0.2380)compared with the control group after 0 hours of CVB3 infection,but decreased significantly after 8hours of CVB3 infection(0.88 ± 0.33 vs 1.15 ± 0.23,P = 0.0074).[Conclusion] Theexpression of AGO1 and AGO3 were significantly up-regulated in the CVB3 treated cells,whileAGO2 and AGO4 mRNA and protein expression levels was no significant changement.AGO1 and AGO3 probably play a vital role in the process of VMC infected by CVB3.The expression of a negative regulator of NF-?B pathway TNFAIP3 was down-regulated.It is speculated that the up-regulation of AGO1 and AGO3 may be related to the down-regulation of TNFAIP3 and all of them may be involved in the process of VMCmyocardial injury.Part ? Molecular Mechanism of Argonaute Protein Involved in Cardiomyocytes Infected with Coxsackie Group B Virus Type 3 Virus[Object] To explore the molecular mechanism of myocardial injury after Coxsackie B group 3 virus(CVB3).[Methods]The male inbred Balb/ c neonatal mice cardiomyocytes infected by CVB3 after 3 days of isolated and cultured.Small interfering RNAs of AGO1 and AGO3 were designed and synthesized,namely siAGO1 and siAGO3,which can knockdown the expression of corresponding genes in cardiomyocytes,and then we detect the expression of TNFAIP3 and p65 protein levels to study the effect of AGO1 and AGO3 on the activity of NF-?B signaling pathway.Then miR-19 a,miR-19 b agonists and miR-19 a,miR-19 b inhibitorswere designed and synthesized,and they were transfected into NIH3T3 cells,cardiomyocytes and siAGO1,siAGO3-treated CVB3-infected cardiomyocytes.Then the expression of TNFAIP3 and miR-19 a and miR-19 b was detected.[Results] After 8 hours of CVB3 infection in cardiomyocytes,the expression of TNFAIP3 was increased.Expression of TNFAIP3 was not changed significantly after knockdown AGO1 and AGO3 by si RNA;the total level of p65 in cardiomyocytes did not been affected,but the level of p65 protein in the nucleus and cytoplasm decreased after knockdown of AGO1 and AGO3.After the transfection of miR-19 a and miR-19 b analogues and inhibitors into NIH3T3 cells 48 h,cell total proteins were extracted for Western blot detection.We found that in the mi R-19 a and miR-19 b analogue transfection group,the level of TNFAIP3 in cardiomyocytes was down-regulated,and TNFAIP3 expression level increased in the miR-19 a and miR-19 b inhibitor transfection group.When the specific base in TNFAIP3-3' UTR region was mutated,miR-19 a and miR-19 b analogues and inhibitors did not significantly modify the expression of TNFAIP3.After the transfection of miR-19 a and miR-19 b analogues and inhibitors into cardiomyocytes cells,miR-19 a and miR-19 b analogues down-regulated the expression of TNFAIP3,and the expression of endogenous TNFAIP3 protein in myocardium was down-regulated,mi R-19 a and miR-19 b inhibitors up-regulated the expression of TNFAIP3.After infected by CVB3 for 8 h,miR-19 a and miR-19 b expression level was not changed in cardiomyocytes,but the activity of luciferase was decreased in the CVB3 infection group compared with the control group,that is to say the expression of TNFAIP3 protein was decreased.There was no significant difference in luciferase activity between the two groups after knockdown of AGO1 and AGO3 by siRNA.[Conclusion](1)Overexpression of AGO1 and AGO3 can inhibit the TNFAIP3 level in cardiomyocytes after CVB3 infection.The expression of TNFAIP3 was not changed after knockdown of AGO1 and AGO3 with small interfering RNA(siAGO1 and siAGO3).(2)Overexpression of AGO1 and AGO3 increased the activity of NF-?B signaling pathway in cardiomyocytes after CVB3 infection,and NF-?B activity was decreased after knockdown of AGO1 and AGO3 with small interfering RNA(siAGO1and siAGO3).(3)miR-19 a and miR-19 b inhibit the exogenous expression of TNFAIP3 in NIH3T3 cells by targeting to the 3'UTR region of TNFAIP3.(4)miR-19 a and mi R-19 b inhibit the endogenous expression by targeting to the3'UTR region of TNFAIP3 in cardiomyocytes.(5)The expression of miR-19 a and mi R-19 b don't have significant difference after CVB3 infection in cardiomyocytes.(6)Overexpression of AGO1 and AGO3 results in down-regulation of TNFAIP3 level through miR-19 a,miR-19 b target to TNFAIP3-3'UTR region in cardiomyocytes. |
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