缺氧预处理诱导大鼠心肌细胞差异表达microRNA的芯片筛选与生物信息学分析

Screening and bioinformatics analysis of differentially expressed miRNAs induced by hypoxia preconditioning in rat cardiomyocytes

目的筛选缺氧预处理(hypoxia preconditioning,HPC)诱导成年大鼠心肌细胞差异表达的microRNA(miRNA),并预测分析miRNA调控的靶基因与功能。方法体外分离培养成年大鼠心室肌细胞,分为对照组(CON)和缺氧预处理组(HPC),CON组细胞正常培养,HPC组细胞经历10 min缺氧和30 min再给氧,提取心肌细胞总RNA,行miRNA芯片筛选,qRT-PCR验证芯片结果。利用软件预测差异表达miRNA调控的靶基因,分析靶基因富集的基因功能(gene ontology,GO)和信号通路(pathway)。结果与CON组相比,HPC可诱导大鼠心肌细胞miRNA表达谱发生明显改变,共有12个miRNA上调,14个miRNA下调(P<0.01,FDR<0.05);选择其中荧光信号值>500的7个miRNA,用于生物信息学分析。qRT-PCR检测miR-133b-5p、miR-664-1-5p、miR-6216水平,变化趋势与芯片结果一致。生物信息学分析显示差异表达miRNA所调控的靶基因功能明显富集于27个GO和6条信号通路。结论 HPC可诱导成年大鼠心肌细胞miRNA表达谱明显改变,这些差异表达miRNA可能通过调控靶基因参与HPC介导的心肌细胞保护作用。

Aim To screen the differentially expressed microRNAs ( miRNAs ) induced by hypoxia precondi-tioning ( HPC ) in adult rat cardiomyocytes, and pre-dict miRNAs-regulated target genes and their func-tions. Methods Cardiomyocytes were isolated from a-dult rat ventricular myocardium and cultured ( in vitro) . The cells were divided into 2 groups: control group ( CON ) and hypoxia preconditioning group ( HPC) . The cardiomyocytes in HPC group were sub-jected to 10 min hypoxia followed by 30 min reoxygen-ation, while the cells in CON group were cultured un-der normal condition. After that, total RNA was ex-tracted and then subjected to miRNA microarray to screen differentially expressed miRNAs. The microar-ray results were further validated by quantitative RT-PCR ( qRT-PCR ) . Bioinformatics analysis was per-formed to predict the miRNAs-regulated target genes and analyze the enriched gene ontology ( GO) and sig-naling pathway ( Pathway) . Results HPC caused sig-nificant changes in miRNAs expression in cardiomyo-cytes as compared to CON group. A total of 12 miR-NAs were up-regulated and 14 miRNAs were down-reg-ulated ( P <0. 01 , FDR <0 . 05 ) . The differentially expressed 7 miRNAs with a fluorescent signal intensity>500 were selected for further bioinformatics analysis. The expression of miR-133b-5p, miR-664-1-5p and miR-6216 detected by qRT-PCR exhibited the similar patterns of up or down regulation to those shown in mi-croarray results. Bioinformatics analysis revealed that miRNAs-regulated target genes were significantly en-riched in 27 GOs and 6 signal pathways. Conclusion <br> The expression profile of miRNAs in rat cardiomyo-cytes is significantly affected by HPC. These differenti-ally expressed miRNAs might participate in HPC-in-duced cardioprotection by regulating their target genes in rat cardiomyocytes.