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)

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).

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.

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.

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. Dual-luciferase 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.