Background <p>Gastroesophageal reflux disease (GERD) is a common digestive disease characterized by impaired esophageal mucosal barrier integrity. Growing evidence indicated that mucosal barrier dysfunction of GERD might be attributed to esophageal inflammation, rather than direct chemical damage of acidic refluxates. However, the potential mechanisms underlying the activation of esophageal inflammation in GERD remain unclear.</p> Methods <p>Patients with GERD symptoms (gastroesophageal reflux disease [GERD] and functional esophageal disorders [FED]) and healthy volunteers were prospectively enrolled. GERD-specific miRNA-mRNA pairs were identified and subsequently validated through histological examination and in vitro experiments. The human esophageal epithelial cell (HEEC) line was used to explore the specific mechanism contributing to the mucosal barrier dysfunction.</p> Results <p>Both miR-146a-5p and its target mRNA, KYNU, were significantly up-regulated in GERD compared with FED/healthy volunteers. In vitro, miR-146a-5p overexpression led to a marked increase in KYNU and interleukin-6 (IL-6) expression, accompanied by a decrease in claudin-4 level and dilated intercellular spaces (DIS). Acid bile salt (ABS) stimulation, mimicking the reflux of GERD, could lead to up-regulation of miR-146a-5p and KYNU in HEECs, which resulted in up-regulation of IL-6 and down-regulation of claudin-4, eventually causing DIS. These effects were mitigated by inhibiting miR-146a-5p or KYNU.</p> Conclusion <p>This study provided novel insights into how acidic refluxates drove inflammation damage to esophageal barrier integrity in GERD. By modulating GERD-specific pair miR-146a-5p-KYNU, acidic refluxates could promote IL-6 secretion, leading to claudin-4 down-regulation and DIS, ultimately impairing esophageal barrier integrity in GERD.</p>