microRNA-146a and Hey2 form a mutual negative feedback loop to regulate the inflammatory response in chronic apical periodontitis

Chronic apical periodontitis (CAP) is defined as chronic inflammation of the dental pulp and root canal system. Porphyromonas endodontalis lipopolysaccharide (P. endodontalis LPS) plays an important role in inducing an inflammatory response in CAP. microRNA-146a (miR-146a) is a key regulator of inflammation and is induced by LPS. Hairy and enhancer-of-split related with YRPW motif 2 (Hey2) has been confirmed to be induced by the Notch signaling pathway, which is involved in tooth development, pulp regeneration, and repair after injury. Our study aimed to investigate the functional role of miR-146a via the targeting of Hey2 in CAP as well as the underlying mechanism. Compared with 13 healthy controls, miR-146a and Hey2 expressions were significantly higher in 20 patients with CAP. In addition, miR-146a, Hey2, interleukin (IL)-6, IL-1 beta, and tumor necrosis factor (TNF)-alpha expressions were significantly increased in MC3T3-E1 cells stimulated with different concentrations (0-20 mu g/mL) of P. endodontalis LPS for different amounts of time (0-48 hours). Moreover, miR-146a, which acts as an anti-inflammatory mediator, negatively regulated the expression of IL-6, IL-1 beta, and TNF-alpha, and Hey2 was confirmed as a target gene of miR-146a by a luciferase reporter assay. Hey2 also negatively regulated miR-146a, IL-6, IL-1 beta, and TNF-alpha expressions, and P. endodontalis LPS strongly induced Hey2 recruitment to the IL-6 promoter (-400 similar to -200 bp). These findings suggest that miR-146a and Hey2 form a mutual negative feedback regulatory loop, demonstrating a novel mechanism that regulates inflammatory responses in CAP.