Role of microRNAs in regulating endothelial cell functions

该研究计划的中心假设是，通过调节内皮细胞粘附受体如E-选择素的表达，microRNAs（miRNAs）是循环细胞与EC粘附的主要调节因子。特别地，我们假设miRNA在反馈回路中起作用以抑制E-选择素的表达。我们将追求三个具体目标来检验我们的假设。 1)明确miRNAs在调节细胞粘附和跨内皮细胞迁移中的作用。在用炎性细胞因子处理以诱导E-选择素的表达之前，将用miRNA的模拟物或抑制剂转染EC。然后，将在静态和动态条件下评价细胞的粘附。此后，将评价miRNA的表达以及它们结合E-选择素mRNA的3 'UTR区的能力。 2)确定触发miRNAs表达的信号通路。将评估已知调节E-选择素表达以响应炎性细胞因子的途径的参与。这些方法将依赖于使用化学抑制剂、显性阴性和siRNA介导的这些途径的蛋白质敲低。 3)探讨miRNAs表达的调控机制。将测定pri-miRNA的转录水平。还将评估前体miRNA的加工和miRNA的稳定性。RNA诱导沉默（RISC）复合物的组成将通过实验室中使用的适当尖端方法确定。磷酸化的RISC组件的作用也将被确定。 总的来说，这一系列的实验将使我们能够第一次建立miRNA和miRNA调节途径如何调节E-选择素的表达，以调节循环细胞与EC的粘附。因此，我们的创新研究计划将为理解miRNAs在细胞-细胞粘附和细胞迁移中的作用开辟新的途径。最终，我们的研究结果将有助于更好地了解炎症，免疫和癌症。

The central hypothesis of this research program is that microRNAs (miRNAs) by regulating the expression of endothelial adhesion receptors such as E-selectin are major regulators of the adhesion of circulating cells to ECs. In particular, we hypothesize that miRNAs act in a feedback loop to repress the expression of E-selectin. We will pursue three specific objectives to test our hypothesis. 1) To ascertain the role of miRNAs in regulating cell adhesion and transendothelial cell migration. ECs will be transfected with mimics or inhibitors of miRNAs before being treated with inflammatory cytokines to induce the expression of E-selectin. Then, the adhesion of cells will be evaluated in static and dynamic conditions. Thereafter, the expression of miRNAs will be evaluated as well as their capacity to bind to the 3'UTR region of E-selectin mRNA. 2) To identify the signaling pathways triggering the expression of miRNAs. The involvement of the pathways known to regulate the expression of E-selectin in response to inflammatory cytokines will be evaluated. The approaches will rely on the use of chemical inhibitors, dominant negatives and siRNA-mediated knockdown of proteins of these pathways. 3) To investigate the mechanisms regulating the expression of miRNAs. The transcription level of pri-miRNAs will be determined. The processing of the pre-miRNAs and the stability of miRNAs will also be evaluated. The composition of the RNA-induced silencing (RISC) complex will be determined by appropriate cutting edge approaches in use in the lab. The role of phosphorylation of the RISC components will also be ascertained. Overall, this series of experiments will allow us to establish for the first time how miRNAs and miRNA-regulating pathways modulate E-selectin expression to modulate the adhesion of circulating cells to ECs. In corollary, our innovative research program will open new avenues in understanding the role of miRNAs in cell-cell adhesion and cell migration. Ultimately, our results will help to better understand inflammation, immunity and cancer.