【作者】 李建华；

【导师】 侯应龙；

【作者基本信息】 山东大学 ， 内科学（心血管病）， 2019， 硕士

【摘要】 研究背景心房颤动（atrial fibrillation,AF）是临床实践中最常见的心律失常之一,其发病率常年居高不下且随年龄的增长而增加,房颤全球患病率约为1%-2%,我国房颤患病率约为0.77%。房颤患者易发脑卒中和心功能衰竭等并发症,具有高致残率、高致死率和治疗效果不佳等特点,其疾病负担亦逐年加重。房颤的治疗主要包括药物治疗、介入治疗和外科迷宫手术,但治疗效果及预后依然不尽人意,这与其复杂的发病机制尚未被完全阐明有关。已有研究证明,心房肌能量代谢重构是房颤发生和维持的重要机制。心房肌能量代谢重构,即心房肌细胞能量代谢紊乱和代谢路径的改变,包括高能磷酸盐代谢的改变、线粒体的功能紊乱及底物利用的转变,导致心房肌生理功能和组织结构的异常改变,从而影响房颤的发生。长链非编码RNA（long non-coding ribonucleic acid,lncRNA）是一组长度超过200 nt,无蛋白编码功能的RNA分子,其在基因转录、蛋白质翻译等多个层面调控基因的表达。已有研究证明,lncRNAs参与多种心脏疾病的发生,如心力衰竭、室间隔缺损、心肌梗塞、房颤电重构及自主神经重构等。LncRNAs是否参与房颤心房肌能量代谢重构,并发挥重要作用,国内外至今尚无系统研究。研究目的本研究通过右心房快速起搏方法构建房颤兔模型,应用高通量测序技术（第二代测序技术）检测对照组和房颤组实验兔右心房肌组织中lncRNAs的差异性表达,并采用生物信息学分析方法,筛选与房颤心房肌能量代谢重构密切相关的目标lncRNA。通过目标lncRNA功能缺失实验,研究其对心房肌能量代谢重构及房颤发生的影响,阐明其分子生物学机制,为房颤的发生提供新的解释。材料与方法1.房颤兔模型构建取12只健康成年新西兰白兔,体质量2.0-2.5kg,雌雄不拘,随机分为对照组（n=6）和房颤组（n=6）。两组实验兔均行心脏起搏器植入术,电极置于右房,房颤组以10 Hz（600次/分）起搏7天构建房颤模型,对照组不予起搏。心脏起搏器植入术前及7天后,分别行心脏电生理检查,包括房颤诱发性和心房有效不应期（atrial effective refractory period,AERP）。2.LncRNAs高通量测序及生信分析TRIzol法提取对照组（n=3）和房颤组（n=3）实验兔右心房肌总RNAs,采用高通量测序技术检测两组实验兔右心房肌组织中lncRNAs的差异性表达,并对显著差异性表达的转录本（differentially expressed transcripts,DETs）进行生物信息学分析,包括:基因本体论（gene ontology,GO）富集分析、京都基因与基因组百科全书（kyoto encyclopedia of genes and genomes,KEGG）通路分析、共表达分析、基因组织特异性表达分析等。筛选出与房颤心房肌能量代谢重构相关的lncRNATCONS₀0016478及其靶基因PGC1-α。3.目标lncRNA作用机制的预测（1）cis-机制筛选距目标lncRNA上下游<10 kb的mRNAs,实时定量聚合酶链式反应法（quantitative real-time polymerase chain reaction,qRT-PCR）检测目标lncRNA及其邻近mRNAs的表达水平,统计学方法分析其表达相关性。（2）trans-机制基于皮尔逊相关系数（pearson correlation coefficient,PCC）>0.85且P<0.05,筛选与目标lncRNA共表达的mRNAs,行GO富集分析与KEGG通路分析,预测目标lncRNA的trans-机制。通过文献检索及qRT-PCR检测,寻找与目标lncRNA有关的靶基因、信号通路及分子机制。4.目标lncRNA敲低实验（1）体外实验构建阴性对照及TCONS₀0016478沉默慢病毒,慢病毒滴度1 ×l09 TU/mL。提取新生乳兔原代心房肌细胞并培养,随机分4组:空白对照组,不感染慢病毒;阴性对照组,感染阴性对照慢病毒;TCONS₀0016478沉默组,感染TCONS₀0016478沉默慢病毒;TCONS₀0016478沉默与PGCl-α过表达共处理组,同时感染TCONS₀0016478沉默慢病毒和PGCl-α过表达慢病毒。培养96 h后收集细胞用于后续实验。（2）在体实验健康成年新西兰白兔18只,雌雄不拘,随机分为假手术组（n=6）,行开胸术但不注射慢病毒;阴性对照组（n=6）,右心房注射阴性对照慢病毒;TCONS₀0016478沉默组（n=6）,右心房注射TCONS₀0016478沉默慢病毒。分别于慢病毒感染前与慢病毒感染7天后检测房颤诱发性和AERP等心脏电生理指标。各组实验兔于感染慢病毒7天后处死,取心房肌组织用于后续实验。5.分子生物学指标检测采用qRT-PCR法检测TCONS₀0016478、靶基因PGC]-α及其下游基因的表达量;Western Blotting法检测靶基因PGC]-α及其下游基因的蛋白质表达水平。6.腺嘌呤核苷酸检测各组实验兔右心房肌组织剪碎、匀浆后提取腺嘌呤核苷酸（ATP、ADP和AMP）,采用高效液相色谱法（high performance liquid chromatography,HPLC）分析心房肌组织中腺嘌呤核苷酸的含量。7.组织特殊染色分析各组实验兔右心房肌组织分别行过碘酸希夫反应（periodic acid schiff,PAS）染色检测糖原沉积,油红O染色检测脂滴沉积。研究结果1.通过右心房快速起搏方法成功建立房颤兔模型,与对照组相比,房颤组AERP明显缩短,房颤诱发率升高。2.测序发现对照及房颤兔右心房肌组织中存在99843个全新lncRNAs转录本,存在差异性表达的有1220个,其中237个表达上调,983个表达下调。3.通过对差异性表达的转录本进行GO富集分析、KEGG通路分析、共表达分析、靶基因预测分析、基因组织特异性表达分析、文献检索等一系列生物信息学方法,筛选出1个与心房肌能量代谢重构相关的全新转录本TCONS₀0016478行进一步研究。4.通过基因序列比对发现,TCONS₀0016478和PGC1-α共同位于第2号染色体,TCONS₀0016478位于PGC1-α基因下游,相距1637 bp（<3kb）。同对照组相比,房颤组实验兔TCONS 00016478与PGC1-α在基因水平表达量均降低,两者呈表达相关性。PGC1-α为TCONS₀0016478靶基因,TCONS₀0016478调控靶基因符合lncRNA调控的cis-机制。5.TCONS₀0016478功能缺失实验证明,细胞水平沉默TCONS₀0016478可下调PGC1-α/PPARγ信号通路能量代谢相关基因的表达,包括PGC1-α、PPARy、GLUT4和CPT1;而沉默TCONS₀0016478的同时过表达PGC1-α,基因PPARγ、GLUT4、CPT1的表达量与对照组相比无统计学差异。研究结果表明,TCONS₀0016478通过调控PGC1-α,间接调节PPARγ、GLUT4、CPT1 的表达。6.动物水平沉默TCONS₀0016478,可下调PGC1-α/PPAR－信号通路能量代谢相关基因的表达,导致糖原沉积、脂滴沉积,腺嘌呤核苷酸合成减少,心房肌细胞能量代谢障碍。慢病毒感染后,TCONS₀0016478沉默组实验兔AERP缩短,且易诱发房颤,而假手术组和阴性对照组AERP和房颤诱发性无统计学差异。研究结论TCONS₀0016478通过调控PGC1-α/PPARγ信号通路影响实验兔心房肌能量代谢重构和房颤的发生。本研究为房颤的发生提供新的解释,为房颤防治提供新的干预靶点。更多还原

【Abstract】 BackgroundAtrial fibrillation（AF）is one of the most common arrhythmias in clinical practice.The morbidity rate of AF is high and increases with age.The total morbidity rate of AF in the world was about 1%-2%,and the number in our country was about 0.77%.AF is associated with stroke and heart failure,resulting in an enormous burden worldwide which is closely related to the high disability and mortality.Treatments for AF mainly include drug therapy,interventional therapy,and Maze procedure.But the therapeutic effect and prognosis of AF is still not satisfactory because of the mechanisms of AF has not been fully elucidated.Previous studies had confirmed that atrial energy metabolic remodeling plays important roles in the initiation and maintenance of AF.Atrial energy metabolic remodeling mainly includes myocardial cell energy metabolism disorder and metabolic pathway changes,for example alterations in adenine nucleotides,mitochondrial function,and a change in substrate.These alterations cause abnormal changes in myocardial physiological function and tissue structure,resulting in the occurrence of AF.LncRNAs are non-protein coding RNAs that longer than 200 nt in length.Previous studies had confirmed that IncRNAs are involved in gene transcription and protein translation.Recent studies revealed that lncRNAs play important roles in cardiac diseases,including heart failure,ventricular septal defect,myocardial infarction,electrical remodeling in AF and autonomic neural remodeling in AF.However,the roles and mechanisms of IncRNAs in atrial energy metabolic remodeling in AF have not been fully illustrated yet.ObjectivesIn this study,right atrial tachypacing was used to establish AF rabbit model;high-throughput sequencing technique（next generation high-throughput sequencing technique）was conducted to detect the IncRNA expression profiles of right atria in non-AF and AF rabbit models;a series of bioinformatics methods were used to to identify the lncRNAs related to atrial energy metabolic remodeling in AF.To investigate the roles of target IncRNA on atrial energy metabolic remodeling and inducibility of AF,we knockdown the target IncRNA.We aimed to clarify its molecular biological mechanism and provide new explanation for the occurrence of AF.Materials and Methods1.Establishment of AF rabbit modelsTwelve healthy adult New Zealand white rabbits,weighing 2.0-2.5 kg,of either sex,were randomly divided into control group（n=6）and AF group（n=6）.Both of the two groups were performed with pacemaker implantation,and the endocardial electrode leads were inserted into the right atria.The AF group underwent continuous right atrial tachypacing（600 beats/min）for one week to establish AF rabbit models,while the control group without pacing.Cardiac electrophysiological tests,including AF inducibility and atrial effective refractory period（AERP）were measured before and one week after.he pacemaker implantation.2.High-throughput sequencing of IncRNAs and bioinformatics analysisTotal RNAs of the right atria from control（n=3）and AF groups（n=3）were extracted with TRIzol reagent.High-throughput sequencing technique was conducted to detect the aberrantly expression profiles of IncRNAs in right atria from control and AF groups.The differentially expressed transcripts（DETs）were analyzed by a series of bioinformatics methods,including Gene Ontology（GO）enrichment analysis,Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway analysis,co-expression genes analysis,tissue specificity analysis,and so on.TCONS₀0016478 and the target gene PGC-1α were identified to play important roles in atrial energy metabolic remodeling during AF.3.Mechanism prediction of the target lncRNA（1）cis-predictionAccording to cis-mechanisms,the nearby mRNAs reside within 10 kb of the target IncRNA were searched.The quantitative real-time polymerase chain reaction（qRT-PCR）was used to show the expression levels of the target IncRNA and nearby mRNAs,and statistical analysis was used to assesse the gene expression correlation.（2）trans-predictionThe co-expressed mRNAs of the target lncRNA were selected based on Pearson correlation coefficient（PCC）>0.85 and P<0.05,then GO enrichment analysis and KEGG pathway analysis were conducted to predict the trans-mechanisms.The target mRNAs,signal pathways and molecular mechanisms of lncRNA were based on qRT-PCR and literature retrieval.4.Loss-of-function of the target lncRNA（1）Infection of lentiviruses in vitroNegative control shRNAs and shRNAs specifically silencing TCONS 00016478 were constructed with a titer of 1×109 TU/mL.The primary atrial myocytes,extracted from the new born rabbits,were cultured and randomly divided into four groups:blank control group,with no infection;negative control group,infected with negative control lentiviruses;lenti-RNAi-TCONS 00016478 group,infected with lentiviruses for silencing TCONS 00016478;co-infected lentiviruses group,infected with lentiviruses for silencing TCONS₀0016478 and lentiviruses for over express PGC-1α.The cells were collected 96 h after infection,and would be used for further experiments.（2）Infection of lentiviruses in vivoEighteen adult New Zealand white rabbits of either sex were randomly divided into the sham group（n=6）,performed by sterile thoracotomy merely;negative control group（n=6）,right atria were infected with negative control lentiviruses;and lenti-RNAi-TCONS 00016478 group（n=6）,right atria were infected with lentiviruses for silencing TCONS 00016478.AF inducibility and AERP were measured after thoracotomy and one week after infection.After one week infection,atrial samples were collected for further experiments.5.Molecular biological assayThe gene expression levels of TCONS 00016478,PGC1-α and the downstream genes were determined by qRT-PCR;the protein expression levels of PGC1-α and the downstream genes were determined by Western Blotting.6.Adenine nucleotides determinationThe atrial tissues were cut into pieces and homogenized,then the adenine nucleotides（ATP,ADP and AMP）were extracted and assessed by high performance liquid chromatography（HPLC）.77.Tissue special stainingThe accumulation of glycogens were assessed by PAS staining.The accumulation of lipid droplets were assessed by Oil Red O staining.Results].The AF rabbit models were successfully established.Compared with the contral group,AERP was shortened in AF group,and the AF inducibility was increased.2.According to the high-throughput RNA-Seq,a total of 99843 new transcripts were detected in the right atria of AF group and control group.There are 1220 differentially expressed transcripts（DETs）,in which 237 DETs were up-regulated and 983 DETs were down-regulated.3.A new transcript TCONS₀0016478 was selected to explore its roles in atrial energy metabolic remodeling after a series of bioinformatics,such as GO enrichment analysis,KEGG pathway analysis,co-expressed genes analysis,target genes prediction,tissue-specific gene expression analysis,literature search,and so on.4.Sequence analysis revealed that TCONS,00016478 and PGC1-α were located on chromosome 2,TCONS₀0016478 was a downstream gene of PGC-1α,the distance was 1637 bp（<3 kb）.Compared with the control group,the gene expression of TCONS₀0016478 and PGC-la were both decreased in AF group,the expression of PGC-la had co-expression correlation with TCONS₀0016478.TCONS₀0016478 was a cis-regulator to regulate the taeget gene PGC-1α.5.Loss-of-function of TCONS₀0016478 verified that silencing TCONS₀0016478 in vitro could inhibit PGC-1α/PPARy pathway genes related to energy metabolism,include PGC1-α,PPARy,GLUT4,and CPT1.Silencing TCONS₀0016478 and overexpressing PGC1-α,the gene expression levels of PPARγ,GLUT4,and CPT1 were not statistically different from the control group.The results indicated that TCONS₀0016478 indirectly regulated the expression of PPARy,GLUT4 and CPT1 by regulating PGC1-α.6.Silencing TCONS₀0016478 in vivo could inhibit PGC-1α/PPARy pathway genes related to energy metabolism,resulting in the glycogens accumulation,lipid droplets accumulation and decrease in adenine nucleotides concentrations.After infection,the AERP was shortened and AF inducibility was increased in TCONS 00016478 silencing group,however,no significant difference in AERP or AF inducibility was found between the sham group and negative control group.ConclusionsTCONS₀0016478 plays important roles in modulating atrial energy metabolic remodeling in rabbits of AF by regulating PGC-1α/PPARγ pathway.Our study provided new explanation for the occurrence of AF and found new targets for prevention/treatment of AF.更多还原