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

组织因子（TF）是血液凝固的起始因子，是心血管疾病和血栓性事件的发病机制之一。选择性剪接导致两种TF亚型的产生。 全长TF是决定血液和血管壁的促凝性的膜结合蛋白。选择性剪接的TF同种型是具有促血管生成特征的可溶性蛋白。最近，我们详细描述了在姜黄素刺激的人内皮细胞中TF-前mRNA选择性剪接的机制。Cdc 2样激酶和DNA拓扑异构酶I通过调节丝氨酸/富含丝氨酸（SR）蛋白的活性来调节丝氨酸诱导的内皮细胞TF亚型表达。这导致TF活性的改变，因此，在确定的流变条件下的内皮细胞的促凝性。MicroRNA（miRNA）是一类短的非编码RNA，是基因表达的重要转录后调节因子。控制内皮细胞中TF同种型应激诱导表达的miRNA尚不清楚。通过利用不同的数据文件库进行计算机分析，以鉴定可能的miRNA，其可能能够调节参与TF-前mRNA的选择性剪接过程的蛋白质的表达。鉴定了几种不同的候选miRNA，其可能有助于TF同种型表达的调节。 此外，我们在第一个主要证据实验中表明，miR-181 b以不同的方式调节两种TF亚型的内皮表达。在本研究中，我们希望研究预先确定的候选miRNAs如何调节TF亚型的表达，特别关注TF的选择性剪接。候选miRNA（如miR-181 b）与相关剪接因子（如Cdc 2样激酶、DNA-拓扑异构酶I或SR-蛋白）的相应靶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）家族效应蛋白的共免疫沉淀分析来表征。此外，将在确定的流变条件下在层流室中离体研究TF亚型表达的miRNA依赖性调节对内皮促凝性的影响。将在患有炎性和缺血性心肌病的患者的外周血（PBMC）的单核细胞中评估候选miRNA的表达模式，以评估它们对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新的见解，心血管止血的中央调节机制，并可能导致确定新的目标，为发展抗血栓形成的策略。

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.