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

Abstract

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.shear stress | endothelial cells | miRNA | nucleolin | AMPK V ascular endothelium responds differentially to pulsatile and oscillatory shear stresses (PS and OS stresses, respectively) which are athero-protective and athero-prone, respectively. Shear stress acting on vascular endothelial cells (ECs) regulates a plethora of microRNAs (miRs), which in turn positively or negatively affect endothelial function. miR maturation is a multistep process involving highly orchestrated events in both the nucleus and cytoplasm. Following transcription in the nucleus, primary miRs (pri-miRs) containing a stem loop structure undergo two sequential cleavage steps initiated by removal of the 3′ and 5′ terminal ends by Drosha, a component of the microprocessor complex. Subsequently, the premiRs are shuttled from the nucleus to the cytoplasm and become truncated, resulting in singlestranded miRs that are 21-23 nucleotides in length. These mature miRs bind to the miR-induced silencing complex (miRISC) containing an argonaute (AGO) protein that targets mRNAs for degradation via miR seed sequence complementarity. In the context of vascular biology, shear stress-regulated miRs modulate EC proliferation, inflammation, nitric oxide (NO) bioavailability, and intercellular communication (1-6). Although the effects of PS and OS on miRs have been well studied, the mechanistic basis for miR processing in ECs responding to distinct shear stress is unknown.Nucleolin (NCL) is a multidomain protein that shuttles between the nucleus and cytoplasm to coordinate various functions (7). Through four tandem nuc...

miR-484: A Potential Biomarker in Health and Disease

“…However, reactive oxygen species (ROS) degradation of BH4 leads to eNOS inactivation causing EC dysfunction. miR-484 and miR-93 in ECs are able to target the seed sequence of the 3’UTR of eNOS and Krüppel-like factor 2 (KLF2) mRNAs and repress protein transcription ( 31 , 55 ). The sustained low expression of miR-484 in ECs under physiological conditions protects against endothelial injury by pulsatile shear (PS) and oscillatory shear (OS).…”

Aptamer-Functionalized Natural Protein-Based Polymers as Innovative Biomaterials

“…One example of functionalized HA-based scaffolds for controlled delivery of therapeutic agents was designed by Wang et al, who developed a biofunctionalized HA microemulsion drug delivery system co-loaded with shikonin and docetaxel drugs to inhibit glioma growth [ 43 ]. Moreover, a single-stranded DNA aptamer (AS1411) was conjugated to specifically drive the dual drug co-loaded HA-based microemulsion to nucleolin, a protein overexpressed on the surface of glioma cell membranes [ 195 , 196 ]. As a result of the AS1411 aptamer, this drug delivery nanosystem decreased transepithelial electrical resistance, thereby increasing the apparent permeability coefficient in an artificial blood–brain barrier (BBB) model and suggesting that the targeted microemulsion was able to penetrate the BBB as a small nanoparticle (30 nm size).…”

Dendrobium catenatum Lindl. Water Extracts Attenuate Atherosclerosis

“…Shear stress, a force between blood flow and blood vessel endothelium for the unit area of the blood vessel wall, is mainly divided into low shear stress (LSS), high shear stress, laminar shear stress, and oscillation shear stress. LSS can induce endothelial cell (EC) dysfunction and oxidative stress, eventually leading to plaque formation [ 8 , 9 ]. LSS also promotes AS by priming ECs for enhanced expression of inflammatory molecules (e.g., intercellular adhesion molecule-1 [ICAM-1] and vascular cell adhesion molecule-1 [VCAM-1]) [ 10 ].…”