大鼠颈动脉球囊损伤模型的竞争性内源RNA调控网络研究

Construction and Analysis of Competitive Endogenous RNA Regulatory Networks in Rat Carotid Balloon Injury Model

目的构建大鼠颈动脉球囊损伤模型的竞争性内源RNA(competing endogenous RNA,ceRNA)调控网络,筛选血管新生内膜形成中的关键调控基因。方法首先建立大鼠颈动脉球囊损伤模型。将12只SD大鼠随机分为2组(每组6只):手术组,利用2F取栓球囊于左颈总动脉行球囊损伤术;假手术组:夹闭左颈总动脉,使血流阻断时间接近球囊损伤术操作时间。术后14 d取颈总动脉血管组织,提取总RNA,进行长链非编码RNA(long non-coding RNA,lncRNA)和信使mRNA(messenger RNA,mRNA)测序。应用生物信息学方法进行差异表达lncRNA和mRNA共表达分析、基因功能(Gene Ontology,GO)和信号通路(Pathway)分析;通过数据库预测微小RNA(microRNA,miRNA)与lncRNA及mRNA的结合关系,并从中筛选已报道在大鼠血管内膜增生过程中有生物学功能的miRNA相关的结合关系,得到最终的ceRNA网络。结果与假手术组相比,手术组鉴定出1731个差异表达的mRNAs和99个差异表达的lncRNAs;GO和Pathway分析显示,差异表达的mRNA主要参与调节免疫反应、细胞迁移、细胞黏附等。ceRNA分析构建了由46个mRNAs、18个lncRNAs和16个miRNAs组成的ceRNA网络,其中AABR07073245.1、Col6a3、AABR07071659.2和AABR07067310.2所介导的ceRNA调控轴可能在大鼠血管新生内膜形成中具有重要调控作用。结论本研究系统分析大鼠颈动脉球囊损伤模型表达谱,构建的ceRNA调控网络可能在大鼠血管内膜增生过程中具有重要调控作用,有望为动脉粥样硬化的防治提供新的靶点与分子标志物。

Objective To screen for key regulatory genes in intima formation,we explored long non-coding RNAs(lncRNAs)and messenger RNAs(mRNAs)expression profiling in a rat carotid balloon injury mo del,and constructed a competitive endogenous RNA(ceRNA)regulatory network.Methods A total of 12 Sprague-Dawley(SD)rats were randomly assigned to two groups:sham-operated group(6 SD rats)was subjected to ischemia through clamping the left common carotid artery;operation group(6 SD rats)underwent the left common carotid arterial injury operation by dilated balloon.Rats were sacrificed and blood vessels were harvested 14 d after operation.All RNAs obtained from the arterial tissue were performed lncRNA/mRNA sequencing using the Illumina platform.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses were performed for differentially expressed mRNAs by bioinformatics methods.Co-expression analyses of differentially expressed lncRNAs and mRNAs were conducted.Cross-talk involving micro-RNAs(miRNAs),lncRNAs and mRNAs was predicted in the databases,and the initial ceRNA network was constructed.The miRNAs which were reported to play roles in the process of intimal hyperplasia in rats were picked out,and the final ceRNA network was obtained.Results There were a total of 1731 differentially expressed mRNAs and 99 differentially expressed lncRNAs between the operation group and the sham-operated group.Differentially expressed mRNAs are mainly involved in regulating immune response,cell migration,and cell adhesion.ceRNA analysis constructed a network consisting of 46 mRNAs,18 lncRNAs,and 16 miRNAs.Further literature search revealed that the ceRNA regulatory axis regulated by AABR07073245.1,Col6a3,AABR07071659.2 and AABR07067310.2 might play an important regulatory role in the neointima formation in rats.Conclusion This study systematically analyzed expression profiles in a rat carotid balloon injury model,and suggested that a ceRNA regulatory network may provide some new targets and molecular markers for the treatment of atherosclerosis.