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Shear stress regulation of miR-93 and miR-484 maturation through nucleolin

Significance This work identifies a mechanism by which atheroprone oscillatory shear and atheroprotective pulsatile shear differentially regulate microRNA (miR) biogenesis in the vascular endothelium. Nonphosphorylated nucleolin facilitates the maturation of miR-93 and miR-484, which in turn target Krüpple-like factor 2 and endothelial nitric oxide synthase mRNAs. The translational significance of these findings is provided by the identification of elevated levels of miR-93 and miR-484 in sera from patients with coronary artery disease. Pulsatile shear (PS) and oscillatory shear (OS) elicit distinct mechanotransduction signals that maintain endothelial homeostasis or induce endothelial dysfunction, respectively. A subset of microRNAs (miRs) in vascular endothelial cells (ECs) are differentially regulated by PS and OS, but the regulation of the miR processing and its implications in EC biology by shear stress are poorly understood. From a systematic in silico analysis for RNA binding proteins that regulate miR processing, we found that nucleolin (NCL) is a major regulator of miR processing in response to OS and essential for the maturation of miR-93 and miR-484 that target mRNAs encoding Krüppel-like factor 2 (KLF2) and endothelial nitric oxide synthase (eNOS). Additionally, anti–miR-93 and anti–miR-484 restore KLF2 and eNOS expression and NO bioavailability in ECs under OS. Analysis of posttranslational modifications of NCL identified that serine 328 (S328) phosphorylation by AMP-activated protein kinase (AMPK) was a major PS-activated event. AMPK phosphorylation of NCL sequesters it in the nucleus, thereby inhibiting miR-93 and miR-484 processing and their subsequent targeting of KLF2 and eNOS mRNA. Elevated levels of miR-93 and miR-484 were found in sera collected from individuals afflicted with coronary artery disease in two cohorts. These findings provide translational relevance of the AMPK–NCL–miR-93/miR-484 axis in miRNA processing in EC health and coronary artery disease.