The role of microRNAs for the regulation of stress-induced tissue factor isoform expression of human endothelial cells and mononuclear leukocytes

microRNA在调节应激诱导的人内皮细胞和单核白细胞组织因子亚型表达中的作用

• 批准号: 243248771
• 负责人: Professorin Dr. Ursula Rauch-Kröhnert
• 金额: --
• 依托单位: Division of Hematology Oncology
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/243248771?language=en
• 关键词: role; microRNAs; regulation; stress; induced

Tissue Factor (TF), the initiator of blood coagulation, is a contributor to the pathogenesis of cardiovascular disease and thrombotic events. Alternative splicing leads to the genesis of two TF isoforms. Full length TF is a membrane-bound protein that determinates the procoagulability of blood and vessel wall. The alternatively spliced TF isoform is a soluble protein with pro-angiogenic characteristics. Recently, we described the mechanism of alternative splicing of TF-pre-mRNA in cytokine-stimulated human endothelial cells in detail. Cdc2-like kinases and DNA-topoisomerase I regulate the cytokine-induced TF isoform expression in endothelial cells by modulating the activity of serin/arginin-rich (SR) proteins. This led to alterations of the TF activity and, thus, the procoagulability of endothelial cells under defined rheological conditions. MicroRNAs (miRNA) are short non-coding RNAs and important post-transcriptional regulators of the gene expression. The miRNAs, that control the stress-induced expression of the TF isoform in endothelial cells, are unknown. In silico analyses were performed by utilizing different data file banks in order to identify possible miRNAs, which might be able to regulate the expression of proteins involved in the alternative splicing process of TF-pre-mRNA. Several different candidate miRNAs were identified that possibly contribute to the regulation of the TF isoform expression. Furthermore, we showed in a first proof-of-principal experiment that miR-181b regulated the endothelial expression of the two TF isoforms in a differential manner. In this study proposal, we want to examine how the pre-identified candidate miRNAs regulate the TF isoform expression with a special focus on the alternative splicing of TF. The interactions between the candidate miRNAs, such as miR-181b, and the respective target-mRNAs of relevant splice factors, such as Cdc2-like Kinases, DNA-topoisomerase I or SR-proteins, will be characterized via interaction studies with the mRNA-target sequences as well as co-immunoprecipitation analyses with effector proteins of the argonaute (Ago) family. Moreover, the effect of the miRNA-dependent regulation of the TF isoform expression on the endothelial procoagulability will be studied ex vivo in a laminar flow chamber under defined rheological conditions. The expression pattern of the candidate miRNAs will be assessed in mononuclear cells of the peripheral blood (PBMC) of patients with inflammatory and ischemic cardiomyopathy with respect to their possible usefulness for estimating the clinical prognosis. In summary, the proposed study will enable us to obtain new insights into central regulatory mechanisms of the cardiovascular hemostasis and will possibly lead to the identification of new targets for the development of anti-thrombotic strategies.

组织因子（TF）是凝血的引发剂，是心血管疾病和血栓事件发病机制的重要因素。选择性剪接导致两种 TF 同工型的产生。 全长 TF 是一种膜结合蛋白，决定血液和血管壁的促凝血性。选择性剪接的 TF 同工型是一种具有促血管生成特性的可溶性蛋白。最近，我们详细描述了细胞因子刺激的人内皮细胞中 TF-pre-mRNA 选择性剪接的机制。 Cdc2 样激酶和 DNA 拓扑异构酶 I 通过调节富含丝氨酸/精氨酸 (SR) 蛋白的活性来调节内皮细胞中细胞因子诱导的 TF 同工型表达。这导致了 TF 活性的改变，从而改变了内皮细胞在特定流变条件下的促凝血能力。 MicroRNA (miRNA) 是短非编码 RNA，是基因表达的重要转录后调节因子。控制内皮细胞中应激诱导的 TF 亚型表达的 miRNA 尚不清楚。通过利用不同的数据文件库进行计算机分析，以确定可能的 miRNA，这些 miRNA 可能能够调节参与 TF-pre-mRNA 选择性剪接过程的蛋白质的表达。鉴定出几种不同的候选 miRNA，它们可能有助于 TF 同工型表达的调节。 此外，我们在第一个原理验证实验中表明，miR-181b 以差异方式调节两种 TF 亚型的内皮表达。在本研究提案中，我们想要研究预先确定的候选 miRNA 如何调节 TF 同工型表达，特别关注 TF 的选择性剪接。候选 miRNA（例如 miR-181b）与相关剪接因子（例如 Cdc2 样激酶、DNA 拓扑异构酶 I 或 SR 蛋白）各自的靶 mRNA 之间的相互作用将通过与 mRNA 靶序列的相互作用研究以及与 argonaute (Ago) 家族效应蛋白的免疫共沉淀分析来表征。此外，将在规定的流变条件下在层流室中离体研究 TF 同工型表达的 miRNA 依赖性调节对内皮促凝血性的影响。将在炎症性和缺血性心肌病患者的外周血（PBMC）的单核细胞中评估候选 miRNA 的表达模式，以确定它们对于估计临床预后的可能有用性。总之，拟议的研究将使我们能够获得对心血管止血的中央调节机制的新见解，并可能导致确定抗血栓策略开发的新靶点。