Background: Disruption of epithelial tight junctions (TJs) promotes loss of polarity and increased invasiveness in gastric cancer. Claudin-18.2 (CLDN18.2), a stomach-specific TJ protein, is frequently downregulated or mislocalized in tumors. However, its functional role remains unclear. This study investigated how CLDN18.2 regulates tight junction integrity and interacts with junction-associated proteins in gastric cancer cells.

Methods: CLDN18.2 expression and subcellular localization in gastric cancer cells (MKN45) and normal gastric epithelial cells (GES-1) were examined using Western blotting and immunofluorescence. CLDN18.2 was silenced using siRNA in MKN45 and SNU-16 cells, and epithelial barrier function was evaluated by transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-Dextran permeability assays. The effects of CLDN18.2 knockdown on tight junction and adherens junction proteins were analyzed by Western blotting. Cell migration and invasion were evaluated using Transwell assays and wound-healing assays in MKN45 cells. Co-immunoprecipitation (Co-IP) was performed to validate CLDN18.2–Zonula occludens-1 (ZO-1) interactions, and rescue experiments with ZO-1 overexpression were performed to determine its ability to restore tight junction integrity following CLDN18.2 silencing. An orthotopic gastric cancer xenograft model was established in BALB/c nude mice using stably transfected MKN45 cells.

Results: CLDN18.2 was highly expressed in MKN45 cells and predominantly localized at the plasma membrane, co-localizing with ZO-1 and occludin. CLDN18.2 knockdown significantly reduced ZO-1, occludin, E-cadherin, and β-catenin expression (p < 0.001), decreased TEER, and increased FITC-Dextran permeability (p < 0.001), while markedly enhancing cell migration and invasion (p < 0.01). Pull-down assays confirmed that CLDN18.2 forms a complex with ZO-1 in an expression-dependent manner. ZO-1 overexpression partially restored tight junction protein levels and barrier function impaired by CLDN18.2 knockdown. In vivo, silencing of CLDN18.2 significantly increased the number of metastatic nodules in the orthotopic xenograft model (p < 0.001). In contrast, overexpression of ZO-1 effectively reversed this pro-metastatic effect and restored junctional protein expression.

Conclusion: CLDN18.2 maintains tight junction integrity in gastric cancer cells through interaction with ZO-1. Its downregulation disrupts cell-cell junctions, weakens epithelial barrier function, and promotes cell migration and invasion, whereas compensatory ZO-1 overexpression can partially reverse these effects. These findings reveal a key regulatory role of CLDN18.2 in gastric cancer progression, offering mechanistic support for CLDN18.2-targeted therapeutic strategies.