Methylation-mediated silencing of ATF3 promotes thyroid cancer progression by regulating prognostic genes in the MAPK and PI3K/AKT pathways

The methylation specific polymerase chain reaction (PCR) and bisulfite sequencing PCR assays were used to identify the methylation status of ATF3 promoter. DNA pull-down combined with mass spectrum assays were conducted to identify TFs binding to ATF3 promoter. The regulatory effects of TFs on ATF3 expression were confirmed by quantitative PCR (qPCR), luciferase reporter assay and chromatin immunoprecipitation (ChIP)-qPCR. Function assays in vitro and xenograft mouse model in vivo were conducted to investigate the function of ATF3 in thyroid cancer. Integrated analysis based on RNA sequencing (RNA-seq), ChIP-seq and CUT&Tag assays were performed to explore the mechanisms underlying the function of ATF3. The associations of ATF3 and its targets with the prognosis of thyroid cancer were analyzed by Kaplan-Meier curves and log-rank tests.