Linlin Hao, Jian Zhang, Qi Qian, Min Jiang, Yu Liu, Yan Geng, Nan Cui, Qi Guo, Jie Guo
5-Methylcytosine (m5C) is a vital RNA modification that regulates mRNA stability, splicing, and nuclear export. As a key m5C reader, ALYREF promotes tumorigenesis across various cancers; however, its precise role in endometrial cancer (EC) remains unknown. ALYREF expression and clinical significance in EC were evaluated using bioinformatics and immunohistochemistry. In vitro and in vivo assays examined its functional roles. Integrated multi-omics (RNA-seq, RIP-seq, and m5C-BS-seq) and rescue assays identified downstream targets. RNA stability, nuclear-cytoplasmic fractionation, site-directed mutagenesis, dual-luciferase, immunofluorescence, and Western blotting were performed to elucidate the underlying mechanisms. ALYREF is significantly upregulated in EC and serves as an independent prognostic factor for poor overall survival. Functional studies demonstrated that ALYREF markedly promotes EC cell proliferation, migration, invasion, and tumor growth. Mechanistically, XRCC6 was identified as a direct functional target. ALYREF enhances XRCC6 mRNA stability in an m5C-dependent manner, subsequently activating canonical Wnt/β-catenin signaling to drive EC progression. Our findings reveal that ALYREF functions as a potential oncogenic driver in EC. The ALYREF-m5C-XRCC6 axis accelerates EC progression via Wnt/β-catenin signaling, highlighting a promising therapeutic target.