MicroRNA-146a-5p alleviates lipopolysaccharide-induced NLRP3 inflammasome injury and pro-inflammatory cytokine production via the regulation of TRAF6 and IRAK1 in human umbilical vein endothelial cells (HUVECs)

Abstract

Background: Microribonucleic acids (miRNAs) have an evident role in regulating endothelial inflammation and dysfunction, which characterizes the early stages of atherosclerosis. The NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome has been reported to contribute to the endothelial inflammatory response that promotes atherosclerosis development and progression.This study sought to investigate the effects of miR-146a-5p on lipopolysaccharide (LPS)-induced NLRP3 inflammasome injury and pro-inflammatory cytokine production in human umbilical vein endothelial cells (HUVECs).Methods: HUVECs were transfected with a miR-146a-5p mimic, small-interfering RNA (siRNA) (si-TRAF6, and si-IRAK1), and were then stimulated with LPS for 24 h. The messenger (mRNA) and the protein levels of p-NF-κB/NF-κB, NLRP3, Caspase-1, pro-inflammatory cytokine [interleukin (IL)-6, IL-1β and tumor necrosis factor alpha (TNF-α)] in the HUVECs were analyzed by quantitative real-time polymerase chain reactions (PCRs) and western blot assays, respectively. The secretion of IL-6 from the cells was detected by enzyme-linked immunoassay (ELISA). Bioinformatic and dual-luciferase reporter assays were performed to identify the targets of miR-146a-5p.Results: LPS promoted pro-inflammatory cytokine expression in a dose-dependent manner and significantly increased the expression levels of p-NF-κB/NF-κB p65, NLRP3, and Caspase-1. After transfection with a miR-146a-5p mimic, or si-TRAF6 or si-IRAK1, we observed that the mRNA and protein levels of NF-κB/p-NF-κB, NLRP3, Caspase-1, and pro-inflammatory cytokine in the HUVECs were all down-regulated, and the secretion of IL-6 from cells declined significantly. After transfection with a miR-146-5p mimic, the expression of TRAF6 and IRAK1 in HUVECs were both down-regulated. Dualluciferase reporter assays confirmed that miR-146-5p directly targets the 3'-untranslated region (3'-UTR) of TRAF6 and IRAK1 to regulate their expression.Conclusions: As a modulator of TRAF6 and IRAK1, miR-146a-5p negatively regulated LPS-induced NF-κB activation and the NLRP3 inflammasome signaling pathway in HUVECs. Thus, miRNA-146a-5p may serve as a potential therapeutic target for atherosclerosis.

miR‑146a‑5p negatively regulates the IL‑1β‑stimulated inflammatory response via downregulation of the IRAK1/TRAF6 signaling pathway in human intestinal epithelial cells

“…IRAK1 and TRAF6, were identified using TargetScan Human database (targetscan.org/vert_80/) as involved in IL-1β signaling. miR-146a-5p directly targets TRAF6 and IRAK1 genes to decrease NF-κB activation and inflammation in human umbilical vein endothelial (48) and bronchial (BEAS-2B) (49) and primary lung epithelial cells (50). In the present study, expression of IRAK1 and TRAF6 was downregulated following incubation with IL-1β.…”

Study on the regulatory effect of Panax notoginseng saponins combined with bone mesenchymal stem cell transplantation on IRAK1/TRAF6-NF-κB pathway in patients with diabetic cutaneous ulcers

“…Previously, we found that miR-146a-5p was lowly expressed in DCU through bioinformatics analysis, and IRAK1 and TRAF6 were binding target genes of miR-146a-5p [ 32 ]. Besides, IRAK1 and TRAF6 were up-regulated in DCU.…”

miR-146a-5p Attenuates Allergic Airway Inflammation by Inhibiting the NLRP3 Inflammasome Activation in Macrophages

“…Meanwhile, overexpression of miR-146a-5p blocked the elevation of NLRP3, ASC, and cas-pase1 proteins in OVA mouse lung tissue. miR-146a-5p attenuates lipopolysaccharide-induced NLRP3 inflammasome damage and pro-inflammatory cytokine production [21]. In addition, NLRP3 was reported to be involved in the regulation of asthma macrophages [6].…”

“…Moreover, miR-146a is mainly involved in regulating inflammation and other functional processes in the innate immune system and is a particularly important negative regulator of nuclear factor κB (NF-κB) signaling [19,20]. Besides, miR-146a-5p negatively regulates LPSinduced NF-κB activation and NLRP3 inflammasome pathways as a regulator of TRAF6 and IRAK1 [21]. However, the mechanism of miR-146a-5p reducing airway inflammation in asthma by regulation of NLRP3 inflammasome activation and macrophage M2 polarization has not been reported at home and abroad.…”