The Regulation Of MiRNA-222 And PTEN By ADAR1 In CVB3-induced Viral Myocarditis

Objective:Viral myocarditis(VMC)is a potentially serious life-threatening cardiovascular disease,which can lead to arrhythmia,cardiac dilation,heart failure or even death in severe cases,as well as the main causes of sudden death in adolescents.Studies have shown that the direct and indirect damage of viruses to cardiomyocytes caused by strong immune response are the main pathophysiological pathways of viral myocarditis,but the more comprehensive and specific pathogenesis and pathogenesis are still remained unclear.MiRNAs are a type of endogenous non-coding small RNAs,which can participate in the pathophysiological process of many diseases by binding to the 3'terminal non-coding sequence of target RNA through base complementary pairing,and further inhibiting protein translation.Many studies have found that miRNAs are closely related to the pathogenesis of viral myocarditis.For example,microRNAs-203 enhances the replication ability of Coxsackievirus B3 by regulating the expression of zinc finger protein-148,and microRNAs-381 enhances the function of cardiomyocytes by regulating cyclooxygenase-2.Adenosine deaminases acting on RNA(ADAR)is a recently discovered as endogenous editing enzyme that can modify microRNAs.On the one hand,it can reprogram the precursor of microRNAs and block their maturation process;on the other hand,it combined with Dicerase to accelerate the maturation process of microRNAs and participated in the formation of gene silencing complexes.In this study,BALB/C mice and myocardial cells were taken as the research objects to explore whether ADAR1 was involved in the regulation of the formation of microRNAs in viral myocarditis,thus playing a role in the pathogenesis,providing theoretical basis for clinical treatment and forensic pathological identification.Methods:In this study,mice were infected with CVB3 by intraperitoneal injection to establish viral myocarditis model,and HE staining was used to identify the infliating the inflammatory cytokines.Western blot(WB)was used to detect the expression of ADAR1 in viral myocarditis,and co-precipitation(CO-IP)to detect the complex of ADAR1 and Dicerase.The expression level of microRNA-222 was examined in viral myocarditis by RT-qPCR.The downstream target protein PTEN(gene of phosphate and tension homology deleted on Chromsome ten,PTEN)was predicted by targetscan and related literature.After up-regulation and down-regulation of microRNA by miRNA-222 mimics and inhibitors,respectively,the expression of PTEN was detected by WB.After the expression of ADAR1p150 was up-regulated and down-regulated by plasmid and siRNA transfection,the expression of microRNA222 and PTEN was detected by RT-q PCR and WB,respectively.Finally,cell counting kit(CCK-8)was used to detect the activity of cardiomyocytes which ADAR1p150 knocked out.Result:(1)Establishment of viral myocarditis modelHE staining showed that the myocardial fibers of mice were wavy after 7 days of virus infection.Inflammatory cells infiltrated mainly by neutrophils in the myocardial interstitium,and the myofibers were broken.(2)The expression of ADAR1 in viral myocarditisThe expression of ADAR1p150 in the heart tissue of mice infected with CVB3 virus was significantly higher than that of the control group,with statistical significance,while the expression of subtype ADAR1p110 was not significantly different from that of the control group.To further verify this result,we detected the expression of ADAR1 in Neonatal rat cardiomyocytes(NRVM),primary cardiac fibroblasts(CF)and H9c2 cell lines of SD rats infected with CVB3 virus by cell culture experiment in vitro.The trend was consistent with that of animal models.(3)Complexes of ADAR1p150 and DicerImmunocoprecipitation analysis showed that ADAR1p150 and Dicer formed complex and directly interacted with each other.(4)The expression and regulation of microRNA222 in viral myocarditisThe results of RT-q PCR showed that the relative expression of microRNA-222 in heart tissue was significantly higher than that in control group,while the relative expression of microRNA(microRNA-221,17,151,432)had no significant difference.The same detection method found that the changes of microRNA-222 in NRVM and CF infected with the virus had the same trend as that in heart tissue.Target scan predictive analysis found that PTEN was the target of microRNA-222.In cardiac myocytes,the expression of microRNA-222 was up-regulated and down-regulated through microRNA mimics and microRNA inhibitor.Results The expression of PTEN decreased with the increase of microRNA-222,whereas it increased.(5)ADAR1p150 regulates the expression of microRNA222 and PTENAfter up-regulation and down-regulation of ADAR1p150 in primary cardiac myocytes of SD rats,WB results showed that the expression of microRNA222 and PTEN had significant changes:after up-regulation of ADAR1p150,the expression of microRNA222 increased and the expression of PTEN decreased;after down-regulation of ADAR1p150,the expression of microRNA222 decreased and the expression of PTEN increased.To further clarify this result,we inhibited the expression of microRNA-222 after H9c2 infection,and found that the down-regulated PTEN showed a trend of recovery.(6)Effect of endogenous ADAR1p150 on myocardial cell viability and apoptosis-related proteinsAfter the inhibition of ADAR1p150 expression in primary myocardial cells of SD rats,the myocardial cells were infected with microviruses(1/2 of the conventional dose,5 times the plurality of infection).After 24 hours and 48 hours,CCK-8 was used to detect the cell viability.The results showed that the cell viability inhibited by ADAR1p150 expression was significantly lower than that of the control group.At the same time,the changes of apoptosis-related proteins were detected by WB,and the surface of Bcl 2 was found.The expression of Bax was higher than that of the control group,suggesting that the apoptotic rate was increased in the experimental group.It was concluded that ADAR1p150 could produce anti-apoptotic protective effect on cardiomyocytes.Conclusion:(1)Up-regulated expression of ADAR1p150 in viral myocarditis.(2)ADAR1p150 and Dicer enzyme form complex to regulate the expression of miRNA-222/PTEN in viral myocarditis.(3)In viral myocarditis,the up-regulation of ADAR1p150 has a protective effect on myocardium.