The focal distribution of atherosclerotic lesions in the arterial tree is attributable to the exposure of the vascular endothelial cells (ECs) to the atheroprone oscillatory shear (OS) at vessel branch points and curvatures, in contrast to the atheroprotective pulsatile shear (PS) at straight parts of the vessel. These two types of flow patterns induce distinct expression profiles of miRNAs (miRs) that differentially regulate the expression of target geness, resulting in distinct functional outcomes. Nucleolin (NCL) is a multi-functional protein that plays critical roles in regulating cell functions through epigenetic, transcriptional, post-transcriptional, and translational regulations. We propose that NCL regulates miRome (miR transcription and maturation) through its interactions with DNA (via g-quadruplex sequences) and RNA (via UCCCGA consensus sequence). Our preliminary results have demonstrated that PS induces the phosphorylation of NCL that leads to an increased binding of NCL to the promoter/enhancer regions of miR23b/27b to increase miR transcription. On the other hand, under OS, the non- phosphorylated NCL binds to miR93/miR484 and Drosha to facilitate miR maturation. These novel findings have led to the hypothesis of this renewal proposal: NCL is differentially regulated by atheroprotective and atheroprone flow patterns to modulate the miRome at transcriptional and maturation levels, thus playing an important role in vascular homeostasis in health and disease. To test this hypothesis, we propose the following four Specific Aims: 1) to examine the molecular mechanisms underlying PS- vs. OS-regulation of NCL in miR transcriptome, 2) to determine the molecular mechanisms underlying PS- vs. OS-regulation of NCL- modulated miR maturation, 3) to integrate the multi-layer regulations of miRome by NCL and the functional consequences under PS and OS with the use of systems approaches, 4) to validate the role of NCL in regulating vascular function in vivo. In the proposed research, we will use a combination of in vitro endothelial cell biology, in vivo mouse models, in silico network construction/data mining, and clinical samples derived from patients with cardiovascular diseases to decipher the mechanisms of flow modulation of NCL-regulation of miRome. The findings will result in novel understanding of the role of NCL in regulating vascular functions in health and disease via miR regulations. !
We propose to use the systems biology approach combining in vitro, in vivo, and in silico experimental procedures, as well as human specimens, to elucidate the flow-regulation of nucleolin to modulate microRNA transcription and maturation, as well as the targeted gene expression in vascular endothelial cells. The resultant mechanistic and regulatory network models will provide critical information on vascular biology in normal and pathophysiological conditions. The study will also provide novel knowledge for the prevention, treatment, and management of vascular diseasses.
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