Li Houwei, Liu Xue, Sun Na, Wang Tianshuo, Zhu Jia, Yang Shuang, Song Xia, Wang Ruishuai, Wang Xinhui, Zhao Yixiu, Zhang Yan
Department of Cardiology at the Second Affiliated Hospital of Harbin Medical University, Harbin, China.
Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics; Key Laboratory of Cardiovascular Medicine Research, Ministry of Education), College of Pharmacy, Harbin Medical University, Harbin, China.
Front Cardiovasc Med. 2021 Jul 7;8:657544. doi: 10.3389/fcvm.2021.657544. eCollection 2021.
Circular non-coding RNA (circRNA) has a variety of biological functions. However, the expression profile and potential effects of circRNA on atherosclerosis (AS) and vascular endothelial injury have not been fully elucidated. This study aims to identify the differentially expressed circRNAs in atherosclerotic aortic vessels and predict their potential functions in endothelial injury. ApoE-/- mice were fed with high-fat diet for 12 weeks to induce AS. Atherosclerotic plaques were evaluated by H&E and Masson staining and immunohistochemistry; differentially expressed circRNAs were detected by Arraystar Circular RNA Microarray and verified by RT-PCR; the potential target mircoRNAs of circRNAs were predicted by miRanda, Tarbase, Targetscan and their expression changes were verified by RT-PCR; the potential target genes of mircoRNAs were predicted by Targetscan and verified by Western blot; the signaling pathways that they might annotate or regulate and their potential functions in vascular endothelial injury were predicted by gene enrichment analysis. Fifty two circRNAs were up-regulated more than twice and 47 circRNAs were down-regulated more than 1.5 times in AS aortic vessels. Mmmu_circRNA_36781 and 37699 were up-regulated both in AS aortic vessels and HO-treated mouse aortic endothelial cells (MAECs). The expression of miR-30d-3p and miR-140-3p, the target microRNA of circRNA_37699 and circRNA_36781, were downregulated both in AS vessels and HO-treated MAECs. On the contrary, MKK6 and TP53RK, the potential target gene of miR-140-3p and miR-30d-3p, were upregulated both in AS aortic roots and HO-treated MAECs. Besides, gene enrichment analysis showed that MAPK and PI3K-AKT signaling pathway were the most potential signaling pathways regulated by the differentially expressed circRNAs in atherosclerosis. Mmu_circRNA_36781 (circRNA ABCA1) and 37699 (circRNA KHDRBS1) were significantly up-regulated in AS aortic vessels and HO-treated MAECs. They have potential regulatory effects on atherosclerosis and vascular endothelial injury by targeting miR-30d-3p-TP53RK and miR-140-3p-MKK6 axis and their downstream signaling pathways.
环状非编码RNA（circRNA）具有多种生物学功能。然而，circRNA在动脉粥样硬化（AS）和血管内皮损伤中的表达谱及潜在作用尚未完全阐明。本研究旨在鉴定动脉粥样硬化主动脉血管中差异表达的circRNA，并预测它们在内皮损伤中的潜在功能。给载脂蛋白E基因敲除（ApoE-/-）小鼠喂食高脂饮食12周以诱导AS。通过苏木精-伊红（H&E）染色、Masson染色和免疫组织化学评估动脉粥样硬化斑块；通过Arraystar环状RNA微阵列检测差异表达的circRNA，并通过逆转录-聚合酶链反应（RT-PCR）进行验证；通过miRanda、Tarbase、Targetscan预测circRNA的潜在靶标微小RNA（miRNA），并通过RT-PCR验证其表达变化；通过Targetscan预测miRNA的潜在靶基因，并通过蛋白质免疫印迹法（Western blot）进行验证；通过基因富集分析预测它们可能注释或调节的信号通路及其在血管内皮损伤中的潜在功能。在AS主动脉血管中，52种circRNA上调超过两倍，47种circRNA下调超过1.5倍。Mmmu_circRNA_36781和37699在AS主动脉血管和血红素加氧酶（HO）处理的小鼠主动脉内皮细胞（MAECs）中均上调。circRNA_37699和circRNA_36781的靶标miRNA miR-30d-3p和miR-140-3p在AS血管和HO处理的MAECs中均下调。相反，miR-140-3p和miR-30d-3p的潜在靶基因MKK6和TP53RK在AS主动脉根部和HO处理的MAECs中均上调。此外，基因富集分析表明，丝裂原活化蛋白激酶（MAPK）和磷脂酰肌醇-3激酶-蛋白激酶B（PI3K-AKT）信号通路是动脉粥样硬化中差异表达的circRNA最可能调节的信号通路。Mmu_circRNA_36781（circRNA ABCA1）和37699（circRNA KHDRBS1）在AS主动脉血管和HO处理的MAECs中显著上调。它们通过靶向miR-30d-3p-TP53RK和miR-140-3p-MKK6轴及其下游信号通路，对动脉粥样硬化和血管内皮损伤具有潜在调节作用。
