Miaomiao Huo ¹ Min Zhang ¹ Jingyao Zhang ¹ Yong Wang ¹ Ting Hu ¹ Tianyu Ma ¹ Yinuo Wang ¹ Baowen Yuan ¹ Hao Qin ¹ Xu Teng ² Hefen Yu ² Wei Huang ^2* Yan Wang ^1*

• ¹ Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
• ² Department of Biochemistry and Molecular Biology, Basic Medical School, Capital Medical University, Beijing, China

Background: Cancers arise from genetic and epigenetic abnormalities that affect oncogenes and tumor suppressor genes, compounded by gene mutations. The N6-methyladenosine (m6A) RNA modification, regulated by methylation regulators, has been implicated in tumor proliferation, differentiation, tumorigenesis, invasion, and metastasis. However, the role of m6A modification patterns in the tumor microenvironment of gastric cancer (GC) remains poorly understood. Materials and methods: In this study, we analyzed m6A modification patterns in 267 GC samples utilizing 31 m6A regulators. Using consensus clustering, we identified two unique subgroups of GC. Patients with GC were segregated into high- and low-infiltration cohorts to evaluate the infiltration proportions of the five prognostically significant immune cell types. Leveraging the differential genes in GC, we identified a “green” module via Weighted Gene Co-expression Network Analysis. A risk prediction model was established using the LASSO regression method. Results: The “green” module was connected to both the m6A RNA methylation cluster and immune infiltration patterns. Based on “Module Membership” and “Gene Significance”, 37 hub genes were identified, and a risk prediction model incorporating nine hub genes was established. Furthermore, methylated RNA immunoprecipitation and RNA Immunoprecipitation assays revealed that YTHDF1 elevated the expression of DNMT3B, which synergistically promoted the initiation and development of GC. We elucidated the molecular mechanism underlying the regulation of DNMT3B by YTHDF1 and explored the crosstalk between m6A and 5mC modification. Conclusion: m6A RNA methylation regulators are instrumental in malignant progression and the dynamics of tumor microenvironment infiltration of GC. Assessing m6A modification patterns and tumor microenvironment infiltration characteristics in patients with GC holds promise as a valuable prognostic biomarker.