低温缺血再灌注心律失常大鼠心肌miRNA表达的变化及靶基因预测

Changes in expression of myocardial miRNA and prediction of target genes in rats with hypothermic ischemia-reperfusion arrhythmia

目的明确低温缺血再灌注心律失常大鼠心肌miRNA表达的变化及其靶基因。方法清洁级健康雄性SD大鼠,2~3月龄,体重300~400 g,麻醉后开胸取心脏,建立离体心脏灌注模型。成功建立离体心脏灌注模型6个,采用随机数字表法分为2组(n=3):对照组(C组)和心脏缺血再灌注组(IR组)。采用全心停灌60 min再灌注30 min的方法制备低温心脏缺血再灌注损伤模型。记录再灌注期间心律失常评分。通过高通量测序筛选出2组心肌表达有显著性差异的miRNA(DEmiRNAs)。利用RNAhybrid和miRanda数据库对DEmiRNAs调节的mRNA行靶基因预测,通过Gene Ontology和KEGG数据库对靶基因进行富集分析,选取与心律失常密切相关且表达水平较高的miRNA进行RT-PCR检测。结果高通量测序结果:与C组比较,IR组有7个miRNA表达有显著性差异(novel-miR-17、novel-miR-19、novel-miR-30、novel-miR-43、rno-miR-122-5p、novel-miR-16和rno-miR-429)。与心律失常关系密切且表达水平较高的miRNA有4个:与C组比较,IR组心肌novel-miR-17、novel-miR-30和rno-miR-122-5p表达上调,rno-miR-429表达下调(P<0.05)。通过miRNA-mRNA相关性分析结果:GJA1基因是novel-miR-17的靶点。结论心肌novel-miR-17可能作用于GJA1基因参与大鼠低温缺血再灌注心律失常的发生。

Objective To determine the changes in the expression of myocardial miRNA and the target genes in the rats with hypothermic ischemia-reperfusion(I/R)arrhythmia.Methods Clean-grade healthy male Sprague-Dawley rats,aged 2-3 months,weighing 300-400 g,were anesthetized,the chest was opened,and the heart was taken to establish an isolated heart perfusion model.Six successfully perfused isolated hearts were divided into 2 groups(n=3 each)using a random number table method:control group(group C)and heart I/R group(IR group).The model of hypothermic global I/R injury was established by interrupting perfusion for 60-min followed by 30-min reperfusion in chloral hydrate-anesthetized rats.The arrhythmia score was recorded during reperfusion.High-throughput sequencing was used to identify the differentially expressed miRNAs in two groups.The RNAhybrid and miRanda databases were used to predict the target genes of mRNA regulated by the differentially expressed miRNAs,and the enrichment for target genes was performed by Gene Ontology and KEGG databases,and the miRNAs closely related to arrhythmia and with higher expression were selected to carry out the real-time polymerase chain reaction detection.Results The results of high-throughput sequencing showed that there were 7 differentially expressed miRNAs(novel-miR-17,novel-miR-19,novel-miR-30,novel-miR-43,rno-miR-122-5p,novel-miR-16 and rno-miR-429)in group IR as compared with group C.There were 4 miRNAs that were closely related to arrhythmia and had higher expression:the expression of novel-miR-17,novel-miR-30 and rno-miR-122-5p was significantly up-regulated,and the expression of rno-miR-429 was down-regulated in group IR when compared with group C(P<0.05).The miRNA-mRNA correlation analysis revealed that GJA1 gene was the target of novel-miR-17.Conclusion Myocardial novel-miR-17 is involved in the occurrence of hypothermic I/R arrhythmia probably by acting on GJA1 gene in rats.