EV-derived miR-1246 promotes lymph node metastasis in esophageal squamous cell carcinoma through targeting the PPP2CB/NF-κB axis

Background Lymph node metastasis (LNM) is a critical factor contributing to poor prognosis in esophageal squamous cell carcinoma (ESCC) patients. Consequently, elucidating its molecular mechanisms and identifying effective therapeutic targets have become a paramount challenge in clinical practice. Methods Plasma samples were systematically collected from healthy individuals, ESCC patients without LNM, and those with LNM. Extracellular vesicles (EVs) were isolated from plasma by size-exclusion chromatography (SEC) and ultracentrifugation (UC) methods, and EV-derived miRNA expression profiles were detected and compared by sequencing. Differentially expressed miR-1246 was selected, and a diagnostic model based on plasma EV-miR-1246 was established to predict LNM. The biological functions of EV-miR-1246 and its target gene PPP2CB, which was identified by mass spectrometry combined with online target prediction methods, in the LNM of ESCC were further investigated using in vitro cellular assays and in vivo animal models. Rescue experiments and NF-κB agonist functional assays were performed to verify the downstream regulatory axis. Results MiR-1246 levels were significantly elevated in plasma extracellular vesicles from ESCC patients with lymph node metastasis. The diagnostic model established based on vesicular miR-1246 showed favorable predictive efficacy for lymph node metastasis status in ESCC. In vitro functional experiments confirmed that vesicular miR-1246 derived from ESCC cells significantly enhanced the migration, invasion, and tube formation capacities of human lymphatic endothelial cells (HLECs). In vivo mouse experiments further confirmed that vesicular miR-1246 derived from ESCC cells significantly promoted lymph node metastasis of esophageal squamous cell carcinoma in mice. Mechanistically, miR-1246 targets and represses PPP2CB expression. Rescue experiments further confirmed that the miR-1246/PPP2CB axis activates NF-κB signaling, thereby promoting lymphatic endothelial activation and lymphangiogenesis. Conclusions This study demonstrates that EV-derived miR-1246 acts as a promising predictive biomarker for lymph-node metastasis in ESCC. Mechanistically, EV-delivered miR-1246 targets PPP2CB to activate NF-κB signaling, thereby driving lymphatic endothelial activation and lymph node metastasis. These findings offer novel diagnostic biomarkers and therapeutic targets for ESCC patients with lymph node metastasis.