Expression Profiles,Functions And Mechanism Of Transfer RNA-Derived Small RNAs In Myocarditis

Myocarditis is the inflammation of the myocardium,and a relatively common but potentially life-threatening disease that affects millions globally,especially pediatric patients and young adults.Myocarditis presents in many different ways,ranging from mild symptoms of chest pain to life-threatening cardiogenic shock and ventricular arrhythmia.Fulminant myocarditis(FM)is a sudden and severe inflammation of the myocardium resulting in myocyte necrosis,edema,and cardiogenic shock,of which the clinical presentations vary widely and may include nonspecific presentation.It is often challenging to diagnose myocarditis due to non-specific symptoms,limitations of current diagnostic strategies,and evolving pathogenesis.Currently,the treatment of patients with myocarditis is mainly supportive and rather empirical,and the core principles of therapies of myocarditis are optimal care of arrhythmia,heart failure and etiology-targeted therapy.There are neither guidelines dedicated to myocarditis nor strategies to tailor therapies to patient’s most favorable benefit.Myocarditis can progress to stable DCM with phenotypic characteristics like dilated chamber,decreased myocardial contractility,stiffened chamber and/or arrhythmia in susceptible individuals.Patients with DCM frequently develop heart failure with high mortality,and studies addressing biopsy-proven myocarditis in patients with DCM report highly variable prevalence ranging from 9%to 30%.As such,there is an urgent need to focus on the etiology and pathogenesis of myocarditis to identify effective diagnostic biomarkers and therapeutic targets for the treatment and prevention of myocarditis.In recent years,extensive efforts have been devoted to the putative functions of transfer RNA-derived small RNAs(tsRNAs)in human diseases.tsRNAs,which were first considered to be random cleavage byproducts of tRNA,are a novel class of small non-coding RNAs and produced through cleavage of mature and precursor tRNAs at various positions.A growing body of evidence has shown that tsRNAs play important roles in cellular homeostasis,adaptation of cellular functions to changing environments,and have a critical modulatory function in human diseases such as inflammation,infection,immunity,metabolism,and tumors.They have also been identified in plasma,which indicates that tsRNAs are stable and may directly affect miscellaneous physiological and pathological processes,which makes them promising candidates for their investigation as biomarkers and therapeutic targets.However,the expression profiles and their potential roles in myocarditis remain elusive.Here,we assessed the expression profiles of circulating tsRNAs in plasma separated from peripheral blood samples of children with FM during both the acute and convalescent phases and age-and sex-matched healthy volunteers by small RNA sequencing,and confirmed the target tsRNA by Taqman probes and quantitative realtime polymerase chain reaction(qRT-PCR)in larger samples.Then,we analyzed its possible functions and mechanism in the pathogenesis of myocarditis induced by lipopolysaccharide(LPS)using fluorescence in situ hybridization(FISH),cell counting kit-8(CCK-8),flow cytometry(FCM),dual-luciferase reporter assay,enzyme-linked immunosorbent assay(ELISA)et al.Taken together,this study clarified the functional mechanism of tiRNA-Gln-TTG-001 in myocarditis,and provide a novel biomarker and a promising therapeutic agent for myocarditis,which would be theoretically and realistically significant for the diagnosis and treatment of myocarditis.This MD thesis is organized in three parts accordingly:Part I Expression profiles,clinical implications and functional analyses of tRNAderived small noncoding RNAs in myocarditisObjectives1.To assess the expression profiles of circulating tsRNAs and confirm the targeted tsRNA in plasma separated from peripheral blood samples of children with FM during both the acute and convalescent phases and age-and sex-matched healthy volunteers and selected the differentially expressed tsRNAs.2.To discuss the relationship between the expression levels of targeted tsRNA(expressed as 2-ΔCT)and the serological or imaging parameters of children with fulminant myocarditis during acute phase,and define its clinical implications.3.To explore the predicted target genes and the possible biological functions of targeted tsRNA in the pathogenesis of myocarditis.Methods1.Small RNA sequencing was adopted to assess the expression profiles of circulating tsRNAs in plasma separated from peripheral blood samples of children with FM during both the acute and convalescent phases and age-and sex-matched healthy volunteers to select candidate tsRNAs.2.Taqman probes and qRT-PCR were used to verify and confirm the targeted tsRNA.3.Regression analyses was applied to analyze correlation of the expression levels of targeted tsRNA(expressed as 2-ΔCG)and the serological or imaging parameters of children with fulminant myocarditis during acute phase,4.Databases such as tRFTar,DianaTools,TargetScan were searched to predict the candidate target genes with binding site of the targeted tsRNA.5.Bioinformatic analysis including GO and KEGG databases were adopted to evaluate the potential biological functions.6.Statistical analysis.Results1.On average,7.02 million trimmed reads were obtained per sequencing library.Under the condition of FC≥1.5 and p<0.05.a total of 13 tsRNAs were differentially expressed in FM-A and CON groups;7 tsRNAs were differentially expressed in paired FM-A and FM-C groups;703 tsRNAs showed no significant trends in paired FM-C and CON groups.2.In line with the small RNA sequence data,tiRNA-Gln-TTG-001 was found to be overexpressed in FM-A group,with a 2.29-fold increase when compared to FM-C group,with a 3.49-fold increase when compared to CON group,and with a 5.38fold increase when compared to HF-CON group(P<0.01).tiRNA-Gln-TTG-001 was confirmed as the targeted tsRNA.3.Levels of tiRNA-Gln-TTG-001 was positively associated with the values of hscTnT,C-reactive protein and procalcitonin(P<0.05),and negatively associated with the values of neutrophil proportion(P<0.05).The correlation coefficient between levels of tiRNA-Gln-TTG-001 and N-Terminal pro-B-type natriuretic peptide(NT-proBNP)/white blood cell counts/erythrocyte sedimentation rate/left ventricular ejection fraction/early Gadolinium enhancement ratio was not statistically significant(P>0.05).4.The potential target gene of tiRNA-Gln-TTG-001 was Chloride intracellular channel 4(CLIC4)with the highest score according to tRFTar,DianaTools,TargetScan.5.Gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)enrichment analyzed that tiRNA-Gln-TTG-001 were found to be related to cell apoptosis,myotube differentiation and metabolism,and it was speculated that the top RAS,mitogen-activated protein kinase(MAPK),PhosphoInositide-3 KinasePI3K(PI3K)-protein serine threonine kinase(AKT)signaling pathways may exert crucial influence on the pathogenesis and progression of FM.The above signaling pathways participate in various processes,such as inflammation,immunity,etc.Conclusions1.There were differentially expressed circulating tsRNAs in children with FM.2.tiRNA-Gln-TTG-001 may be involved in the initiation and progression of FM,and may be disease-specific.3.tiRNA-Gln-TTG-001 may take part in the myocardial injury and inflammation.4.tiRNA-Gln-TTG-001 may regulate myocardial injury and inflammation by targeting CLIC4.5.tiRNA-Gln-TTG-001 may be associated with the apoptosis,differentiation,and abnormal metabolism of cardiomyocytes,and had impact on inflammation and immunity.Part Ⅱ Functions of tiRNA-Gln-TTG-001 in regulating lipopolysaccharideinduced myocardial injury and inflammationObjectives1.To detect the production and secretion of tiRNA-Gln-TTG-001.2.To determine the intracellular localization of tiRNA-Gln-TTG-001.3.To confirm the establishment of lipopolysaccharide(LPS)-induced AC 16 human cardiomyocyte cell line(AC 16)inflammatory injury model.4.To explore the expression and functions of tiRNA-Gln-TTG-001 in LPS-induced AC 16 cell inflammatory injury model.Methods1.qRT-PCR were used to detect the expression levels of tiRNA-Gln-TTG-001 from AC 16 and cell culture supernatant.2.Cyanine 3-(Cy3-)labelled tiRNA-Gln-TTG-001 probes were constructed to determine the intracellular localization of tiRNA-Gln-TTG-001 by FISH.3.tiRNA-Gln-TTG-001 mimics/inhibitor was constructed to overexpress and knockdown the expression of tiRNA-Gln-TTG-001.4.Lipofectamine 3000 was adopted to transfect tiRNA-Gln-TTG-001 mimics/inhibitor and their negative control.5.CCK-8 was used to determine the viability of AC 16.6.Annexin V-phycoerythrin(PE)/7-aminoactinomycin D(AAD)staining and flow cytometry was performed to measure the apoptosis and necrosis of AC 16.7.qRT-PCR and ELISA was used to detect the expression levels of inflammatory factors.8.ELISA was used to detect the expression levels of factors associated with cardiac injury and function.9.Statistical analysis.Results1.tiRNA-Gln-TTG-001 can be detected both in cardiomyocytes and cell culture supernatants.2.The red fluorescent Cy3-labelled probe was detectable both in the cytoplasm and nucleus,3.Toll-like receptor 4(TLR4),which serves a key function in LPS-induced inflammation as the receptor of LPS,and related factors were expressed in AC 16.4.In LPS-induced AC 16 cell inflammatory injury model,it was found that the cell viability decreased significantly(P<0.01),apoptosis and necrosis increased significantly(P<0.01),the messenger RNA(mRNA)expression levels of inflammatory factors,interleukin(IL)-1β,IL-18 increased significantly(P<0.05),and the concentration of IL-1β,-6,-18,creatine kinase-MB(CKMB),cardiac troponin(cTnT)increased significantly(P<0.01),whereas,the concentration of NT-proBNP did not change significantly(P=0.8726).5.The expression levels of tiRNA-Gln-TTG-001 from AC 16 and cell supernatant increased significantly(P<0.05),and the expression levels of tiRNA-Gln-TTG001 from AC 16 were associated with the initiation and progression of myocarditis.6.Compared with LPS-induced AC 16,it was found that,in AC 16 transfected with tiRNA-Gln-TTG-001 mimics,the cell viability decreased significantly(P<0.01),apoptosis and necrosis increased significantly(P<0.01),the mRNA expression levels of inflammatory factors,IL-1β,IL-18 increased significantly(P<0.05),and the concentration of IL-1β,IL-6,IL-18,CKMB,cTnT increased significantly(P<0.05),whereas,the concentration of NT-proBNP did not change significantly(P=0.91).7.Compared with LPS-induced AC16,it was found that,in AC16 transfected with tiRNA-Gln-TTG-001 inhibitor,the cell viability increased significantly(P<0.01),apoptosis and necrosis decreased significantly(P<0.01),the mRNA expression levels of inflammatory factors,IL-1β,IL-6,IL-18 decreased significantly(P<0.05),and the concentration of IL-1β,IL-6,IL-18,CKMB,cTnT decreased significantly(P<0.01),whereas,the concentration of NT-proBNP did not change significantly(P=0.4946).Conclusions1.tiRNA-Gln-TTG-001 is synthesized and secreted by cardiomyocytes.2.tiRNA-Gln-TTG-001 is located both in the cytoplasm and nucleus.3.LPS-induced AC 16 models can be established to mimic myocarditis in vitro.4.tiRNA-Gln-TTG-001 may aggravate the myocardial injury and inflammation induced by LPS.Part Ⅲ Mechanism of tiRNA-Gln-TTG-001 in aggravating lipopolysaccharideinduced myocardial injury and inflammationObjectives1.To detect the expression levels of CLIC4 in LPS-induced AC 16 and AC 16 transfected with tiRNA-Gln-TTG-001 mimics or inhibitor.2.To analyze the intracellular localization of CLIC4.3.To detect the binding site between 3’-untranslated region(3’-UTR)of CLIC4 mRNA and tiRNA-Gln-TTG-001.4.To explore the role of tiRNA-Gln-TTG-001 in myocarditis induced by LPS.Methods1.qRT-PCR were used to detect the expression levels of CLIC4 from AC 16 and AC 16 transfected with tiRNA-Gln-TTG-001 mimics or inhibitor.2.Cy3-labelled tiRNA-Gln-TTG-001 probes and FAM-labelled CLIC4 probes were constructed to determine the intracellular localization of CLIC4 and the colocation between CLIC4 and tiRNA-Gln-TTG-001 by FISH.3.The 3’-UTR of CLIC4 mRNA combined with tiRNA-Gln-TTG-001 was verified by dual-luciferase reporter assays.4.CLIC4 small interfering RNA(siRNA)and CLIC4 overexpression plasmid was constructed to knockdown or overexpress the expression of CLIC4 respectively.5.Lipofectamine 3000 was adopted to co-transfect tiRNA-Gln-TTG-001 mimics+CLIC4 siRNA,and tiRNA-Gln-TTG-001 inhibitor+CLIC4 overexpression plasmid.6.CCK-8 was used to determine the viability of AC 16.7.Annexin V-PE/7-AAD staining and flow cytometry was performed to measure the apoptosis and necrosis of AC 16.8.qRT-PCR and ELISA was used to detect the expression levels of inflammatory factors.9.ELISA was used to detect the expression levels of factors associated with cardiovascular diseases.10.Statistical analysis.Results1.The expression levels of CLIC4 from LPS-induced AC 16 and AC 16 transfected with tiRNA-Gln-TTG-001 mimics increased significantly(P<0.05),whereas the expression levels of CLIC4 from LPS-induced AC 16 transfected with tiRNA-GlnTTG-001 inhibitor decreased significantly(P<0.05).2.The green fluorescent FAM-labelled probe was detectable both in the cytoplasm and nucleus,and overlapped with red fluorescent Cy3-labelled probe.3.Dual luciferase assays revealed that tiRNA-Gln-TTG-001 mimics increased the fluorescence value of the luciferase reporter gene in the group transfected with the wild-type vector of 3’-UTR of CLIC4 mRNA(P<0.05),while the fluorescence value of the luciferase reporter gene in the group transfected with the mutant-type vector of 3’-UTR of CLIC4 mRNA stayed same.4.Compared with LPS-induced AC 16 transfected with tiRNA-Gln-TTG-001 mimics,it was found that,in LPS-induced AC16 co-transfected with tiRNA-Gln-TTG-001 mimics+CLIC4 siRNA,the expression level of CLIC4 decreased significantly(P<0.05),the cell viability increased significantly(P<0.01),apoptosis and necrosis decreased significantly(P<0.01),the mRNA expression levels of IL-1βdecreased significantly(P<0.05),and the concentration of IL-1β,IL-6,IL-18,CKMB,cTnT decreased significantly(P<0.01),whereas,the concentration of NTproBNP did not change significantly(P=0.8174).5.Compared with LPS-induced AC 16 transfected with tiRNA-Gln-TTG-001 inhibitor,it was found that,in LPS-induced AC 16 co-transfected with tiRNAGln-TTG-001 inhibitor and CLIC4 overexpression plasmid,the expression level of CLIC4 increased significantly(P<0.05),the cell viability decreased significantly(P<0.01),apoptosis and necrosis increased significantly(P<0.01),the mRNA expression levels of inflammatory factors,IL-1β,IL-6,IL-18 increased significantly(P<0.05),and the concentration of IL-1β,IL-6,IL-18,CKMB,cTnT increased significantly(P<0.01),whereas,the concentration of NT-proBNP did not change significantly(P=0.9464).Conclusions1.Temporal consistency exists between CLIC4 and tiRNA-Gln-TTG-001.2.Spatial consistency exists between CLIC4 and tiRNA-Gln-TTG-001.3.Structure complementarity exists between CLIC4 and tiRNA-Gln-TTG-001.4.tiRNA-Gln-TTG-001 may aggravate the myocardial injury and inflammation by upregulating CLIC4.