【作者】 王静；

【导师】 韩波；

【作者基本信息】 山东大学 ， 儿科学（专业学位）， 2022， 博士

【摘要】 研究背景心肌炎是好发于儿童及青壮年且存在死亡风险的非缺血性炎性心肌病变。由于缺乏特异性的诊断标记物及根治性的治疗方案,心肌炎的早期诊断和治疗被公认是世界范围内极具挑战性的临床难题。由于心肌炎的病因和发病机制尚未完全阐明,迄今临床一线治疗方案仍然以经验性对症支持治疗为主,无法从根本上逆转疾病的病理生理过程,高达9%-30%的儿童期心肌炎可进展为扩张型心肌病。深入研究心肌炎的病因和发病机制,探寻特异的诊断标记物和精准的治疗靶点,是提高临床诊疗水平和改善患者生活质量及预后的关键。随着分子生物学技术的发展和测序技术的改进,非编码RNA(noncoding RNA,ncRNA)已成为剖析疾病发病机制的关键突破口。转运RNA(transfer RNA,tRNA)来源的小非编码 RNA(tRNA-derived small noncoding RNA,tsRNA)是多种核酸酶剪切tRNA或tRNA前体所产生的具有调节功能的小非编码RNA,分为 tRNA 衍生片段(tRNA-derived fragment,tRF)和 tRNA 半分子(tRNA halves,tRH)两大类。其中哺乳动物的tRH也被称为tRNA来源应激诱导的小RNA(tRNA-derived stress-induced small RNA,tiRNA)。tsRNA结构稳定,保守性强,表达具有组织和时空特异性,可以调节基因的转录和翻译,影响表观遗传过程,对炎症、免疫、代谢、肿瘤等起关键调控作用,并且可以稳定地存在于体液,其中血浆tsRNA的表达量与疾病相关的组织细胞病变程度具有相关性,有望应用于液体活检,为揭示分子生物学功能提供新线索。目前国内外尚没有tsRNA在心肌炎中的表达、功能及其机制的相关研究。本课题首先应用高通量小RNA测序技术筛查与暴发性心肌炎(fulminant myocarditis,FM)相关的tsRNA。继而扩大样本量,借助Taqman荧光探针、实时荧光定量聚合酶链式反应(real-time quantitative reverse transcription polymerase chain reaction,qRT-PCR)明确目标tsRNA,并借助临床指标及生物信息学对其临床意义和生物学功能进行初步评估。进而借助荧光原位杂交(fluorescence in situ hybridization,FISH)、细胞计数试剂-8 法(cell counting kit-8,CCK-8)、Annexin V-藻红蛋白(phycoerythrin,PE)/7-氨基放线菌素 D(7-aminoactinomycin D,7-AAD)双染法流式细胞术、双荧光素酶报告基因检测实验、酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)等探索目标tsRNA在脂多糖(lipopolysaccharide,LPS)诱导的人心肌细胞炎症模型中的功能和机制,为心肌炎的早期诊断和治疗提供理论依据和新的靶点。本课题内容相应地分为三部分:第一部分tsRNA在儿童暴发性心肌炎中的表达、临床意义及生物学功能研究目的1.筛选并验证在FM急性期(FM-A)、FM恢复期(FM-C)及正常健康儿童(CON)血浆中存在差异表达的tsRNA。2.探讨目标tsRNA在FM急性期的表达量与临床评估心肌炎的常用指标之间的相关性,初步评估其临床意义。3.预测目标tsRNA的作用靶点及潜在的生物学功能。研究方法1.应用高通量小RNA测序技术检测FM-A组、FM-C组及CON组外周血浆tsRNA的表达谱,筛查与心肌炎发生发展相关且存在差异表达的候选 tsRNA。2.扩大FM-A组、FM-C组及CON组的样本量,并设置心力衰竭疾病对照(HF-CON)组,应用Taqman荧光探针及qRT-PCR技术对候选tsRNA进行验证,确定目标tsRNA。3.应用相关性分析评估目标tsRNA在FM急性期的表达量与临床评估心肌炎的常用指标之间的关系。4.借助tRFTar、DianaTools、TargetScan等数据库预测与目标tsRNA存在结合位点的候选靶基因。5.应用生物信息学方法,借助基因本体数据库(gene ontology,GO)及京都基因和基因组百科全书数据库(Kyoto encyclopedia of genes and genomes,KEGG)的功能分析评估目标tsRNA的潜在生物学功能。6.统计学分析。研究结果1.高通量小RNA测序结果显示:每个测序文库获得平均702万个去掉接头之后的序列。以差异倍数≥1.5以及P值<0.05为显著性差异表达,FM-A组和FM-C组共有7个存在差异表达的tsRNA,FM-A组和CON组共有13个存在差异表达的tsRNA,FM-C组和CON组则有703个不存在差异的tsRNA。2.qRT-PCR结果显示:tiRNA-Gln-TTG-001在FM-A组的表达量最高,较FM-C组上调2.29倍,较CON组上调3.49倍,较HF-CON组上调5.38 倍(P<0.01),故 tiRNA-Gln-TTG-001 被确定为目标 tsRNA。3.tiRNA-Gln-TTG-001在FM急性期的相对表达量(2-ACT)与超敏肌钙蛋白T、C-反应蛋白、降钙素原呈正相关(P<0.05),与中性粒细胞比值呈负相关(P<0.05),与氨基末端脑钠肽前体水平、白细胞计数、血沉、左室射血分数、早期钆强化率不存在相关性(P>0.05)。4.tRFTar、DianaTools、TargetScan等数据库对氯化物细胞内通道蛋白4(Chloride intracellular channel 4,CLIC4)做出的预测评分最高。5.GO功能分析提示tiRNA-Gln-TTG-001与细胞凋亡、肌管分化和代谢等有关。KEGG代谢通路分析提示tiRNA-Gln-TTG-001的靶标可能涉及RAS、丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)、磷酸肌醇三激酶(PhosphoInositide-3 Kinase,PI3K)-蛋白质丝氨酸苏氨酸激酶(protein serine threonine kinase,AKT)等多条信号通路,涵盖炎症和免疫等过程。结论1.在FM和健康儿童的血浆中存在差异表达的tsRNA。2.tiRNA-Gln-TTG-001可能与心肌炎的发生发展具有相关性,且存在疾病特异性。3.tiRNA-Gln-TTG-001可能与心肌的炎症损伤程度具有相关性。4.tiRNA-Gln-TTG-001可能通过靶向CLIC4在心肌炎中发挥作用。5.tiRNA-Gln-TTG-001可能与心肌细胞凋亡有关,可能通过调节炎症和免疫反应发挥作用。第二部分tiRNA-Gln-TTG-001在脂多糖诱导的AC16人心肌细胞炎症损伤的功能研究研究目的1.探索AC16人心肌细胞系(AC16)是否可以合成及分泌tiRNA-Gln-TTG-001。2.探索tiRNA-Gln-TTG-001在AC16的胞内分布情况。3.探索LPS是否可以诱导AC16建立细胞炎症模型。4.探索tiRNA-Gln-TTG-001在心肌细胞炎症模型中的表达及功能。研究方法1.应用qRT-PCR技术检测AC16及其上清液tiRNA-Gln-TTG-001的表达情况。2.构建花青素荧光染料(Cyanine 3,Cy3)标记的tiRNA-Gln-TTG-001探针,应用FISH技术对tiRNA-Gln-TTG-001在AC16的胞内分布进行定位。3.构建 tiRNA-Gln-TTG-001 mimics/inhibitor 以分别过表达/敲降 tiRNA-Gln-TTG-001。4.应用 lipofectamine 3000 转染 tiRNA-Gln-TTG-001 mimics/inhibitor。5.CCK-8确定细胞活性。6.Annexin V-PE/7-AAD双染法流式细胞术检测细胞凋亡和坏死。7.qRT-PCR及ELISA法确定炎症因子表达量的变化。8.ELISA法确定心肌损伤标记物及心功能标记物表达量的变化。9.统计学分析。研究结果1.AC16细胞及细胞上清液内均可检测到tiRNA-Gln-TTG-001。2.与Cy3标记的tiRNA-Gln-TTG-001探针杂交的AC16的细胞核和细胞质内均可探测到红色荧光。3.qRT-PCR证实AC16存在LPS受体同时也是炎症信号受体的Toll样受体4(Toll-like receptor 4,TLR4)及其相关因子,具备LPS诱导建立细胞炎症模型的必要条件。4.在LPS刺激下,AC16的细胞活力显著降低(P<0.01),细胞凋亡和坏死显著增多(P<0.01),炎症因子白介素(interleukin,IL)-1β、IL-18在信使RNA(messenger RNA,mRNA)水平的表达量显著增高(P<0.05),细胞上清液中炎症因子IL-1β、IL-6、IL-18和心肌损伤标记物肌酸激酶同工酶MB(creatine kinase-MB,CKMB)、肌钙蛋白T(cardiac troponin,cTnT)的表达量显著增高(P<0.01),心功能标记物氨基末端 B 型脑利钠肽前体(N-terminal pro-B-type natriuretic peptide,NT-proBNP)的表达量无显著性差异(P=0.8726)。5.在LPS刺激下,AC16细胞及细胞上清液tiRNA-Gln-TTG-001的表达量均显著增高(P<0.05),其中细胞tiRNA-Gln-TTG-001的表达量与AC16炎症损伤的发生发展存在时间相关性。6.相较于未转染的AC16,转染tiRNA-Gln-TTG-001 mimics的AC16的细胞活力显著降低(P<0.01),细胞凋亡和坏死显著增多(P<0.01),炎症因子IL-1β、IL-18在mRNA水平的表达量显著增高(P<0.05),细胞上清液中炎症因子IL-1β、IL-6、IL-18及心肌损伤标记物CKMB、cTnT的表达量显著增高(P<0.01),心功能标记物NT-proBNP的表达量无显著性差异(P=0.91)。7.相较于未转染的 AC16,转染 tiRNA-Gln-TTG-001 inhibitor 的 AC16 的细胞活力显著改善(P<0.01),细胞凋亡和坏死显著减少(P<0.01),炎症因子IL-1β、IL-6、IL-18在mRNA水平的表达量显著降低(P<0.05),细胞上清液中炎症因子IL-1β、IL-6、IL-18及心肌损伤标记物CKMB、CTnT的表达量显著降低(P<0.01),心功能标记物NT-proBNP的表达量无显著性差异(P=0.4946)。结论1.AC16 可以生成和分泌 tiRNA-Gln-TTG-001。2.tiRNA-Gln-TTG-001分布于AC16的细胞核和细胞质。3.LPS可以诱导AC16建立细胞炎症模型。4.tiRNA-Gln-TTG-001可加重LPS诱导的AC16炎症损伤的程度。第三部分tiRNA-Gln-TTG-001加重脂多糖诱导的AC16人心肌细胞炎症损伤的机制研究研究目的1.探索CLIC4在心肌细胞炎症模型中的表达量,以及不同程度表达量的tiRNA-Gln-TTG-001 对 CLIC4 表达量的影响。2.探索CLIC4在AC16的胞内分布情况,以及CLIC4与tiRNA-Gln-TTG-001的空间关系。3.探索 tiRNA-Gln-TTG-001 是否与 CLIC4 mRNA 的 3’-非编码区域(3’-untranslated region,3’-UTR)存在结合位点。4.探索tiRNA-Gln-TTG-001加重LPS诱导的AC16炎症损伤的机制。研究方法1.应用qRT-PCR技术检测接受LPS刺激的未转染/转染tiRNA-Gln-TTG-001 mimics/inhibitor 的 AC16 中 CLIC4 的表达量。2.构建Cy3标记的tiRNA-Gln-TTG-001探针,FAM标记的CLIC4探针,应用FISH技术对CLIC4在AC16的胞内分布,以及CLIC4与tiRNA-Gln-TTG-001的分布关系进行定位检测。3.构建荧光素酶质粒CLIC4 mRNA的3’-UTR过表达组、CLIC4 mRNA的3’-UTR突变组、CLIC4 mRNA的3’-UTR阴性对照组,应用双荧光素酶报告基因检测实验验证tiRNA-Gln-TTG-001与CLIC4 mRNA的3’-UTR是否存在结合位点。4.构建 CLIC4 小干扰 RNA(small interfering RNA,siRNA)及过表达CLIC4的质粒。5.应用 lipofectamine 3000 分别转染 tiRNA-Gln-TTG-001 mimics+CLIC4 siRNA,以及 tiRNA-Gln-TTG-001 inhibitor+过表达 CLIC4 质粒。6.CCK-8确定细胞活性。7.Annexin V-PE/7-AAD双染法流式细胞术检测细胞凋亡和坏死。8.qRT-PCR及ELISA法确定炎症因子表达量的变化。9.ELISA法确定心肌损伤标记物及心功能标记物表达量的变化。10.统计学分析。研究结果1.qRT-PCR结果显示:在LPS刺激下,AC16中CLIC4的表达量显著增高(P<0.05)。相较于未转染的AC16,过表达tiRNA-Gln-TTG-001的AC16中CLIC4的表达量显著升高(P<0.05),敲降tiRNA-Gln-TTG-001的AC16中CLIC4的表达量显著降低(P<0.05)。2.与FAM标记的CLIC4探针杂交的AC16的细胞核和细胞质均可探测到绿色荧光,同时与Cy3标记的tiRNA-Gln-TTG-001探针和FAM标记的CLIC4探针杂交的AC16的细胞核和细胞质内可探测到红色及绿色荧光,且二者可重叠。3.双荧光素酶报告基因检测实验显示:tiRNA-Gln-TTG-001 mimics+CLIC4 mRNA的3’-UTR过表达组的荧光比值显著增高(P<0.05),即tiRNA-Gln-TTG-001与CLIC4 mRNA的3’-UTR存在固定互补的结合位点,且tiRNA-Gln-TTG-001存在激活CLIC4的潜力。4.在LPS刺激下,相较于单独转染tiRNA-Gln-TTG-001 mimics的AC16,转染 tiRNA-Gln-TTG-001 mimics+CLIC4 siRNA 的 AC16 中 CLIC4 的表达量显著降低(P<0.05),细胞活力显著改善(P<0.01),细胞凋亡和坏死显著减少(P<0.01),炎症因子IL-1β在mRNA水平的表达量显著降低(P<0.05),细胞上清液炎症因子IL-1β、IL-6、IL-18及心肌损伤标记物CKMB、CTnT的表达量显著降低(P<0.01),心功能标记物NT-proBNP的表达量无显著性差异(P=0.8174)。5.在 LPS 刺激下,相较于单独转染 tiRNA-Gln-TTG-001 inhibitor 的 AC16,转染 tiRNA-Gln-TTG-001 inhibitor+过表达 CLIC4 质粒的 AC16 中CLIC4的表达量显著增高(P<0.05),细胞活力显著降低(P<0.01),细胞凋亡和坏死显著增高(P<0.01),炎症因子IL-1β、IL-6、IL-18在mRNA水平的表达量显著增高(P<0.05),细胞上清液炎症因子IL-1β、IL-6、IL-18及心肌损伤标记物CKMB、CTnT的表达量显著增高(P<0.01),心功能标记物NT-proBNP的表达量无显著性差异(P=0.9464)。结论1.CLIC4与tiRNA-Gln-TTG-001的表达存在时间同步性。2.CLIC4与tiRNA-Gln-TTG-001的分布存在空间同轨性。3.CLIC4与tiRNA-Gln-TTG-001的结合位点存在结构固定性和互补性。4.tiRNA-Gln-TTG-001通过上调CLIC4加重心肌炎症损伤。更多还原

【Abstract】 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.更多还原