After double verification with TBS and RT-qPCR, we confirmed that four genes are regulated by DNA methylation: ADAP1, GUCA2A, IL22RA1 and MISP. Current research suggests that the pathogenesis of NEC is mainly due to overactivation of immune inflammation centered on TLR4, dysbiosis of the intestinal microbiota, and severe damage to the intestinal villus structure caused by mesenteric vasculature ischemia, leading to irreversible intestinal barrier damage [33]. This evidence concerns the gene MISP and necrotizing enterocolitis.