低温缺血再灌注心律失常大鼠心室肌lncRNAs表达谱的变化

Changes in lncRNAs expression profile in ventricular myocardium of rats with arrhythmia induced by hypothermic ischemia-reperfusion

目的观察低温缺血再灌注心律失常大鼠心室肌长链非编码RNAs(lncRNAs)表达谱的变化。方法清洁级健康雄性SD大鼠,3~4月龄,体重320~390 g,成功制备离体心脏低温缺血再灌注模型16个,采用随机数字表法分为2组(n=8):对照组(C组)和低温缺血再灌注损伤组(I/R组)。采用全心停灌60 min再灌注30 min的方法制备心脏低温缺血再灌注损伤模型。记录再灌注期间心律失常的类型、频率及持续时间,根据室性心律失常评分将I/R组大鼠分为低危组(I/R-L组)和高危组(I/R-H组)。再灌注结束后取左心室心肌组织进行高通量测序,筛选差异表达的lncRNAs,采用qRT-PCR法随机挑选4个显著差异表达的lncRNAs进行验证,并采用生物信息学方法预测其靶基因,利用Gene Ontology、KEGG通路等数据库对差异表达lncRNAs的靶基因进行富集分析。结果与C组比较,I/R-L组表达上调的lncRNAs有124个,下调的lncRNAs有137个,I/R-H组上调的lncRNAs有100个,下调的lncRNAs有139个。与I/R-L组比较,I/R-H组表达上调的lncRNAs有121个,下调的lncRNAs有118个。3组间表达量变化绝对值≥2且显著差异表达(P<0.01)的lncRNAs有68个。其中,与C组比较,I/R-L组表达显著上调的lncRNAs有40个,表达显著下调的有55个,I/R-H组表达显著上调的lncRNAs有42个,表达显著下调的有48个。与I/R-L组比较,I/R-H组显著上调的lncRNAs有52个,下调的有40个。随机选取4个lncRNAs (MSTRG.136297.1、MSTRG.81170.3、MSTRG.60110.2和MSTRG.97622.63)进行qRT-PCR验证,证实测序结果可靠。这些差异表达lncRNAs的靶基因参与调控的与再灌注心律失常相关的生物过程有17个、KEGG通路有9个,且细胞内钙离子浓度的正向调节和心肌细胞肾上腺素能信号通路分别为差异表达lncRNAs的靶基因富集程度最高的生物学过程和KEGG通路。结论全心低温缺血再灌注后,大鼠心室肌lncRNAs表达谱发生了显著性改变,这些显著差异表达的lncRNAs可能主要通过调控钙离子跨膜转运及心肌细胞中的肾上腺素能信号通路,从而导致再灌注心律失常的发生。

Objective To observe the changes in long non-coding RNAs(lncRNAs)expression profiles in ventricular myocardium of rats with arrhythmia induced by hypothermic ischemia-reperfusion(I/R).Methods Clean-grade healthy male Sprague-Dawley rats,aged 3-4 months,weighing 320-390 g,were used in this study.Sixteen hypothermic I/R models of isolated hearts were successfully established and divided into 2 groups(n=8 each)by a random number table method:control group(group C)and hypothermic I/R group(group I/R).Hypothermic I/R injury models were established by globally stopping perfusion for 60 min followed by reperfusion for 30 min.The type,frequency,and duration of arrhythmia during reperfusion were recorded.The rats in group I/R were further divided into low-risk I/R group(group I/R-L)and high-risk I/R group(group I/R-H)according to the score for ventricular arrhythmia.After the end of reperfusion,the myocardium in the left ventricle was sampled for high-throughput sequencing to screen the aberrantly expressed lncRNAs.The quantitative reverse transcription polymerase chain reaction was used to select 4 aberrantly expressed lncRNAs randomly for verification,bioinformatics method was used to predict the target genes,and databases such as Gene Ontology and KEGG pathway were used to analyze the enrichment of target genes of aberrantly expressed lncRNAs.Results Compared with group C,there were 124 up-regulated lncRNAs and 137 down-regulated lncRNAs in group I/R-L,and 100 up-regulated lncRNAs and 139 down-regulated lncRNAs in group I/R-H.Compared with group I/R-L,there were 121 up-regulated lncRNAs and 118 down-regulated lncRNAs in group I/R-H.There were 68 significantly aberrantly expressed lncRNAs(P<0.01)with a fold-change not lower than two among three groups.Compared with group C,there were 40 lncRNAs with significantly up-regulated expression and 55 lncRNAs with down-regulated expression in I/R-L group,and 42 lncRNAs with significantly up-regulated expression,and 48 lncRNAs with down-regulated expression in I/R-H group.Compared with group I/R-L,there were 52 lncRNAs with significantly up-regulated expression and 40 lncRNAs with down-regulated expression in group I/R-H.Four lncRNAs(MSTRG.136297.1,MSTRG.81170.3,MSTRG.60110.2 and MSTRG.97622.63)were randomly selected for verification using quantitative reverse transcription polymerase chain reaction,confirming that the sequencing results were reliable.The target genes of the aberrantly expressed lncRNAs were found to participate in the regulation of 17 biological processes and 9 KEGG pathways related to arrhythmia induced by I/R.In addition,the positive regulation of intracellular calcium ion concentrations and adrenergic signal transduction in cardiomyocytes were the biological processes with the highest enrichment of target genes of aberrantly expressed lncRNAs and the KEGG pathway,respectively.Conclusion The expression profile of lncRNAs in ventricular myocardium has changed significantly after global hypothermic I/R.These significantly aberrantly expressed lncRNAs may lead to reperfusion arrhythmia mainly by regulating transmembrane transport of calcium ions and adrenergic signal transduction in cardiomyocytes of rats.