心肌肥厚小鼠心肌组织中环状RNA的表达谱分析

Analysis on the expression profile of circRNAs in hypertrophic myocardium mice

目的探索心肌肥厚时环状RNA(circRNA)差异表达的情况及其影响的病理生理进程。方法SPF级C57BL/6J雄性小鼠6只,8~10周龄,按照随机数字表法分为主动脉弓缩窄(TAC)组(n=3)和假手术组(n=3)。通过TAC术建立心肌肥厚小鼠模型。术后4周,采用高通量测序分析检测TAC组与假手术组小鼠左心室心肌组织差异表达的circRNA,并采用R语言软件对circRNA进行主成分分析。通过GO和KEGG 2个数据库进行富集分析推测差异表达circRNA来源基因的基本功能及其参与的生物学通路。采用实时荧光定量聚合酶链反应验证测序结果中差异表达的circRNA。结合差异表达的circRNA和TargetScan预测的微小RNA(miRNA)位点,应用Cytoscape软件构建circRNA-miRNA网络图以预测他们的相互作用。结果对TAC组和假手术组小鼠6个样本中检测到的4580个circRNA进行主成分分析,R语言软件结果提示前3个主成分,即第1、2和3主成分的方差贡献率分别为91.01%、3.19%和2.01%,三者累计方差贡献率为96.21%。在差异表达的circRNA中,TAC组小鼠6个(19%)表达上调、25个(81%)表达下调。GO分析结果表明差异表达的circRNA与心肌肥厚的发生发展密切相关,KEGG通路分析提示低表达的circRNA参与了肌动蛋白细胞骨架的调节。在31个差异表达的circRNA中挑选出15个进行实时荧光定量聚合酶链反应验证,结果显示其中8个circRNA与测序结果一致。circRNA-miRNA共表达网络分析结果显示chr11:65218529-65233184-与mmu-miRNA-30e-3p、mmu-miRNA-30a-3p等相互作用。结论心肌肥厚的小鼠心肌组织中circRNA的表达发生了改变。circRNA可能与相应的miRNA相互作用,通过自噬相关的细胞肥大通路或凋亡等病理表型影响心肌肥厚的发生发展。

Objective To explore the differential expression of circRNAs and their potential impact on the pathophysiological process in cardiac hypertrophy.Methods Six SPF C57BL/6J male mice,aged 8 to 10 weeks,were randomly divided into transverse aortic constriction(TAC)group(n=3)or sham operation(sham)group(n=3)according to random number table method.TAC mouse model was used to induce cardiac hypertrophy.Four weeks after surgery,high-throughput sequencing analysis was performed to detect differentially expressed circRNA in left myocardial tissues of mice between TAC group and sham group,and principal component analysis of circRNA was performed by R language software.Enrichment analysis was performed by GO and KEGG databases to predict the basic functions of differentially expressed circRNA-derived genes and their biological pathways.The differentially expressed circRNAs in the sequencing results were verified by real-time fluorescence quantitative polymerase chain reaction.Cytoscape software was used to construct circRNA-microRNA(miRNA)network maps to predict their interactions by combining differentially expressed circRNA and TargetScan predicted miRNA sites.Results Principal component analysis was performed on 4580 circRNAs detected from 6 samples of mice in TAC group and sham group.The results of R language software indicated that the variance contribution rate of the first 3 principal components,namely the first,second and third principal components,was 91.01%,3.19%and 2.01%,respectively,and the cumulative variance contribution rate of the 3 components was 96.21%.Among the differentially expressed circRNAs,6(19%)were up-regulated and 25(81%)were down-regulated in the TAC group.GO analysis showed that differentially expressed circRNA was closely related to the occurrence and development of cardiac hypertrophy,and KEGG pathway analysis suggested that downregulated circRNA expression was involved in the regulation of actin cytoskeleton.Fifteen out of the 31 differentially expressed circRNAs were selected for real-time fluorescence quantitative polymerase chain reaction verification,and the results showed that 8 circRNAs were consistent with sequencing results.circRNA-miRNA co-expression network analysis results showed that chr11:65218529-65233184-interacts with mmu-miRNA-30e-3p and mmu-miRNA-30a-3p.Conclusions The differential expression of circRNA in hypertrophic myocardium mice is evidenced in TAC mouse model.circRNA may interact with the corresponding miRNA to influence the occurrence and development of cardiac hypertrophy through autophagy-related cellular hypertrophy pathway or apoptosis-related pathological phenotypes.