DNA-mediated ZNF649 downregulation modulates the Hedgehog signaling pathway to reduce the sensitivity of gastric cardia cancer to 5-FU.

Gastric cardia adenocarcinoma (GCA), an aggressive gastric cancer with rising incidence and poor prognosis, often requires chemotherapy and surgery. This study aimed to explore the regulatory mechanism of ZNF649 on 5-FU sensitivity in GCA. ZNF649 expression in GCA was analyzed using The Cancer Genome Atlas (TCGA) database. The relationship between ZNF649 expression and methylation level was analyzed using MethylMix and MethPrimer. GCA data was used to predict ZNF649-related signaling pathways via gene set enrichment analysis (GSEA). qPCR, western blot, and immunohistochemistry were used to detect ZNF649 expression at mRNA and protein levels. MSP was used to detect ZNF649 methylation in clinical samples. Cell viability and apoptosis were assessed by CCK8 and flow cytometry. ZNF649 was downregulated in GCA tissues and cells, negatively correlating with DNA methylation in its promoter. Overexpressing ZNF649 increased GCA cell sensitivity to 5-FU, lowering IC50 values and enhancing apoptosis. ZNF649 negatively regulated the Hedgehog pathway. Knocking down ZNF649 activated Hedgehog signaling, reducing 5-FU sensitivity. DNA methylation silences ZNF649, activating the Hedgehog pathway and weakening GCA cell sensitivity to 5-FU. Targeting ZNF649 and the Hedgehog pathway may overcome 5-FU resistance in GCA patients.