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.

Keywords: AMPK; endothelial cells; miRNA; nucleolin; shear stress.

Fig. 1.

AMPK phosphorylation of NCL. (A) miR binding protein ranking based on the number of putative targeted miRs. (B) Functional domains and AMPK phosphorylation consensus sequences on human, mouse, and rat NCL. (C) In vitro kinase assay demonstrating the phosphorylation of recombinant NCL by AMPK. (D) Isoelectric focusing of NCL isolated from AMPK^+/+ and AMPK^−/− MEF cells. (E) In vitro kinase assay using recombinant AMPK and NCL peptides (11-mers) illustrated in B. (F) Peptide competition assay against SAMS peptide and increasing concentrations of S328A 11-mer peptide. (G) Immunoblot with p-Serine antibody for FLAG-NCL immunoprecipitated from HUVEC treated with AICAR for 10 min. (H and I) Immunoblotting of lysates from HUVECs under PS and OS as well as TA and AA. *P < 0.05. Data are mean ± SEM from three independent experiments.

Fig. 2.

NCL regulation of miR processing. (A) Immunoblot showing the nuclear and cytoplasmic fractionation of NCL in HUVECs treated with or without AICAR, control siRNA (siCtrl), and siAMPK. (B and C) NCL was immunoprecipitated from HUVECs subjected to OS or PS or transfected with NCL S328A or S328D. Immunoblotting was then performed with anti-Drosha and anti-NCL. (D) The NCL binding sequence on miR-93 and miR-484 target sequence for human, mouse, and rat. (E) The number of species (i.e., human, mouse, rat, and so forth) that have predicted interaction between NCL and the indicated miRNAs (17 for premiR-93 and 10 for premiR-484). *P < 0.05. Data are mean ± SEM from three independent experiments (A–C).

Fig. 3.

NCL regulates miR-93 and miR-484 processing. (A) Levels of miR-93-3p, miR-93-5p, and miR-484 in HUVECs transfected with control or siAMPK and then subjected to PS or OS for 3 h. (B) miR levels in HUVECs transfected with NCL S328, S328A, or S328D. (C) NCL was immunoprecipitated from HUVECs transfected with siCtrl or siAMPK and subjected to PS or OS. Levels of premiR-93 and premiR-484 bound to the immunoprecipitated NCL were measured. (D) AGO2 was immunoprecipitated from HUVECs transfected with NCL S328, S328A, or S328D. The levels of AGO2-associated miR-93-3p, miR-93-5p, and miR-484 were measured. (E) Levels of miR-93-3p, miR-93-5p, and miR-484 in the TA of EC-AMPKα2^+/+ or EC-AMPKα2^−/− mice. (F) NCL was immunoprecipitated from the TA or AA from AMPKα2^+/+ or AMPKα2^−/− mice. The NCL-bound miR-93 and miR-484 were then measured. *P < 0.05. Data are mean ± SEM from three independent experiments.

Fig. 4.

NCL-miR-93 and NCL-miR4-84 down-regulates KLF2 and eNOS. (A and B) RNA-seq datasets from OS/PS experiments (18) cross-referenced to those of miR-93 or miR-484 overexpression datasets (GSE86497 and GSE66844). Points in dark blue indicate down-regulated genes in both OS/PS and miR-93 or miR-484 overexpression. Points in blue indicate transcripts down-regulated by miR-93 or miR-484 and OS/PS while points in red indicate eNOS and KLF2. (C) KLF2 and eNOS mRNA levels in HUVECs overexpressing premiR-93 or premiR-484. (D and E) HUVECs were transfected with NCL S328A or S328D in combination with Ctrl siRNA, anti–miR-93, or anti–miR-484. The levels of KLF2 and eNOS mRNA and those bound with AGO2 were measured. (F) Immunoblotting of cell lysates from HUVECs transfected with NCL S328, S328A, or S328D and in the presence or absence of AICAR. (G) NO bioavailability in HUVECs transfected with NCL S328A or S328D in combination with anti–miR-93 or anti–miR-484. (H) KLF2 and eNOS mRNA levels in TA of EC-AMPKα2^+/+ or EC-AMPKα2^−/− mice. *P < 0.05. Data are mean ± SEM from three independent experiments.

Fig. 5.

Higher miR-93 and miR-484 levels in CAD patients. Sera were isolated from patients with CAD (n = 56) or HCs (n = 10) in the discovery cohort (A, C, and E) and the validation cohort (n = 32 and 31, respectively in B, D, and F) and the levels of miR-93-3p, miR-93-5p, and miR-484 were measured. Gray boxes represent the mean and the first and third quantiles; *P < 0.05 (G) Correlation among levels of miR-93-3p, miR-93-5p, and miR-484 were found in combined CAD and control samples from both cohorts. (H) Graphical abstract of the PS and OS regulation of the NCL-miR93/484 axis via AMPK phosphorylation of NCL, which results in EC homeostasis versus dysfunction.