Digestive and liver disease : official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver · 2021
EZH2-mediated suppression of CLDN1 leads to barrier dysfunction in PPI-refractory gastroesophageal reflux disease.
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Ma Teng, Gu Jie, Zhao Ye, Li Su, Zou Duowu, Ge Di
Аннотация
BACKGROUND: PPI-refractory gastroesophageal reflux disease (RGERD) is characterized as the existence of reflux symptoms resistant to optimized PPI treatment. Alleviated mucosal integrity has been regarded as one of the mechanisms of RGERD.
METHODS: RNA sequencing analysis and GSEA were performed. Human biopsy samples, cell lines, and rat models were recruited. Trans-epithelial electrical resistance (TEER) was tested and a FITC-dextran flux assay was performed to detect barrier permeability. Tissue morphology was evaluated using HE staining, while gene expression was measured by qRT-PCR, western blotting, flow cytometry, immunofluorescence, immunohistochemistry, and chromatin immunoprecipitation (ChIP) analysis.
RESULTS: The tight junction protein Claudin-1 is significantly weakened in the RGERD epithelium, while levels of EZH2-mediated H3K27me3 were increased. Forced EZH2 expression in epithelial cells led to H3K27me3 accumulation and Claudin-1 suppression, which consequently caused epithelial barrier dysfunction. Notably, studies on esophagogastroduodenal anastomosis (EGDA) rat models showed the attenuation of Claudin-1 level and barrier function could be rescued by an Ezh2 inhibitor GSK126. ChIP analysis followed by qPCR (ChIP-qPCR) revealed H3K27me3 suppressed CLDN1 via accumulating at the TSS area.
CONCLUSION: For the first time, we explored the attenuated tight junction of RGERD, demonstrating a potential underlying mechanism that EZH2-mediated H3K27me3 could impair esophageal epithelial barrier function by suppressing the transcription of CLDN1.
METHODS: RNA sequencing analysis and GSEA were performed. Human biopsy samples, cell lines, and rat models were recruited. Trans-epithelial electrical resistance (TEER) was tested and a FITC-dextran flux assay was performed to detect barrier permeability. Tissue morphology was evaluated using HE staining, while gene expression was measured by qRT-PCR, western blotting, flow cytometry, immunofluorescence, immunohistochemistry, and chromatin immunoprecipitation (ChIP) analysis.
RESULTS: The tight junction protein Claudin-1 is significantly weakened in the RGERD epithelium, while levels of EZH2-mediated H3K27me3 were increased. Forced EZH2 expression in epithelial cells led to H3K27me3 accumulation and Claudin-1 suppression, which consequently caused epithelial barrier dysfunction. Notably, studies on esophagogastroduodenal anastomosis (EGDA) rat models showed the attenuation of Claudin-1 level and barrier function could be rescued by an Ezh2 inhibitor GSK126. ChIP analysis followed by qPCR (ChIP-qPCR) revealed H3K27me3 suppressed CLDN1 via accumulating at the TSS area.
CONCLUSION: For the first time, we explored the attenuated tight junction of RGERD, demonstrating a potential underlying mechanism that EZH2-mediated H3K27me3 could impair esophageal epithelial barrier function by suppressing the transcription of CLDN1.