Huang Zhuhui, Winata William Adiwignya, Zhang Kui, Zhao Yang, Li Yang, Zhou Ning, Zhou Shaoyou, Fu Wei, Qiao Bokang, Li Guoqi, Shao Yihui, Zheng Jubing, Dong Ran
Department of Cardiac Surgery, Beijing Anzhen Hospital, Capital Medical University, Beijing Institute of Heart, Lung and Blood Vessel Diseases, Beijing 100029, China.
Key Laboratory of Remodeling-Related Cardiovascular Diseases, Capital Medical University, Ministry of Education and Beijing Anzhen Hospital, Beijing Institute of Heart, Lung and Blood Vessel Diseases, Beijing 100029, China.
Cardiol Res Pract. 2020 Feb 14;2020:1481937. doi: 10.1155/2020/1481937. eCollection 2020.
Numerous studies have highlighted that long noncoding RNA (lncRNA) can indirectly regulate the expression of mRNAs by binding to microRNA (miRNA). LncRNA-associated ceRNA networks play a vital role in the initiation and progression of several pathological mechanisms. However, the lncRNA-miRNA-mRNA ceRNA network in endothelial cells under cyclic stretch is seldom studied.
The miRNA, mRNA, and lncRNA expression profiles of 6 human umbilical vein endothelial cells (HUVECs) under circumferential stress were obtained by next-generation sequencing (NGS). We identified the differential expression of miRNAs, mRNAs, and lncRNAs using the R software package GDCRNATools. Cytoscape was adopted to construct a lncRNA-miRNA-mRNA ceRNA network. In addition, through GO and KEGG pathway annotations, we analyzed gene functions and their related pathways. We also adopted ELISA and TUNEL to investigate the effect of si-NEAT1 on endothelial inflammation and apoptosis.
We recognized a total of 32978 lncRNAs, 1046 miRNAs, and 31958 mRNAs in 6 samples; among them, 155 different expressed lncRNAs, 74 different expressed miRNAs, and 960 different mRNAs were adopted. Based on the established theory, the ceRNA network was composed of 13 lncRNAs, 44 miRNAs, and 115 mRNAs. We constructed and visualized a lncRNA-miRNA-mRNA network, and the top 20 nodes are identified after calculating their degrees. The nodes with most degrees in three kinds of RNAs are hsa-miR-4739, NEAT1, and MAP3K2. Functional analysis showed that different biological processes enriched in biological regulation, response to stimulus and cell communication. Pathway analysis was mainly enriched in longevity regulating, cell cycle, mTOR, and FoxO signaling pathway. Circumferential stress can significantly downregulate NEAT1, and after transducing si-NEAT1 for 24 h, inflammatory cytokine IL-6 and MCP-1 were significantly increased; furthermore, fewer TUNEL-positive cells were found in the si-NEAT1 treated group.
The establishing of a ceRNA network can help further understand the mechanism of vein graft failure. Our data demonstrated that NEAT1 may be a core factor among the mechanical stress factors and that cyclic stress can significantly reduce expression of NEAT1, give rise to inflammation in the early stage of endothelial dysfunction, and promote EC apoptosis, which may play an essential role in vein graft failure.
众多研究强调长链非编码RNA（lncRNA）可通过与微小RNA（miRNA）结合间接调控mRNA的表达。lncRNA相关的竞争性内源RNA（ceRNA）网络在多种病理机制的起始和进展中发挥着至关重要的作用。然而，循环拉伸下内皮细胞中的lncRNA-miRNA-mRNA ceRNA网络鲜有研究。
通过下一代测序（NGS）获得6个人脐静脉内皮细胞（HUVECs）在周向应力作用下的miRNA、mRNA和lncRNA表达谱。我们使用R软件包GDCRNATools鉴定miRNA、mRNA和lncRNA的差异表达。采用Cytoscape构建lncRNA-miRNA-mRNA ceRNA网络。此外，通过基因本体（GO）和京都基因与基因组百科全书（KEGG）通路注释，我们分析了基因功能及其相关通路。我们还采用酶联免疫吸附测定（ELISA）和末端脱氧核苷酸转移酶介导的缺口末端标记法（TUNEL）研究小干扰RNA-NEAT1（si-NEAT1）对内皮炎症和凋亡的影响。
我们在6个样本中共识别出32978个lncRNA、1046个miRNA和31958个mRNA；其中，采用了155个差异表达的lncRNA、74个差异表达的miRNA和960个差异表达的mRNA。基于既定理论，ceRNA网络由13个lncRNA、44个miRNA和115个mRNA组成。我们构建并可视化了lncRNA-miRNA-mRNA网络，并在计算其度数后确定了前20个节点。三种RNA中度数最高节点分别是hsa-miR-4739、NEAT1和丝裂原活化蛋白激酶激酶2（MAP3K2）。功能分析表明，不同的生物学过程富集于生物调控、对刺激的反应和细胞通讯。通路分析主要富集于寿命调节、细胞周期、哺乳动物雷帕霉素靶蛋白（mTOR）和叉头框蛋白O（FoxO）信号通路。周向应力可显著下调NEAT1，转导si-NEAT1 24小时后，炎性细胞因子白细胞介素-6（IL-6）和单核细胞趋化蛋白-1（MCP-1）显著增加；此外，在si-NEAT1处理组中发现的TUNEL阳性细胞较少。
ceRNA网络的建立有助于进一步了解静脉移植物失败的机制。我们的数据表明，NEAT1可能是机械应力因素中的核心因子，循环应力可显著降低NEAT1的表达，在内皮功能障碍早期引发炎症，并促进内皮细胞凋亡，这可能在静脉移植物失败中起重要作用。
