Methylation changes of gallbladder DNA during the formation of gallstones.

OBJECTIVE: To investigate the genome-wide methylation changes in gallbladder tissues during gallstone formation, as well as alterations in key signaling pathways and associated gene expression. METHODS: A combined methylation and transcriptomic analysis was performed on gallbladder tissues from gallstone model mice and normal control mice using reduced representation bisulfite sequencing (RRBS) and RNA high-throughput sequencing. Key candidate genes were validated using qRT-PCR, Western blotting, and immunohistochemistry. Cross-species validation was conducted using human gallbladder transcriptomic data from GEO datasets. RESULTS: Integrated RRBS and RNA-seq analysis revealed that abnormal DNA methylation may contribute to gallstone formation by dysregulating gene expression in pathways associated with gallbladder dysfunction. A total of 97 differentially methylated and expressed genes exhibiting significant inverse correlations between methylation and expression levels were identified. The top six genes with the strongest correlations were experimentally validated. Analysis of human gallstone samples identified partial overlapping differentially expressed genes with the mouse model. CONCLUSION: This study demonstrates the potential roles of DNA methylation and gene expression changes in gallbladder tissue during the formation of gallstones. These findings provide a new perspective for further understanding the causes of gallstones and searching for possible clinical therapies.