ABSTRACT: Background: 5-Methylcytosine (m5C) is an important RNA modification that participates in multiple aspects of mRNA metabolism, including stability, splicing, and nuclear export. Through these processes, m5C regulates key biological processes such as cellular growth, proliferation, and apoptosis. ALYREF, a principal m5C reader protein, is frequently dysregulated in various malignancies. By specifically recognizing m5C-modified sites on mRNA, ALYREF regulates RNA metabolism and facilitates tumor cell proliferation, migration, and invasion. However, its specific function and molecular mechanisms in endometrial cancer (EC) remain poorly understood. Methods: The expression of ALYREF in EC was analyzed using bioinformatics approaches, RT-PCR, Western blotting, and immunohistochemistry. In vitro and in vivo experiments were performed to investigate the biological role of ALYREF. RNA sequencing, RNA immunoprecipitation sequencing, m5C bisulfite sequencing, and rescue experiments were conducted to identify the downstream target transcripts of ALYREF. RNA stability assays, nuclear-cytoplasmic fractionation, site-directed mutagenesis, and dual-luciferase reporter assays were employed to elucidate the molecular mechanisms by which ALYREF regulates its downstream target gene, XRCC6. Furthermore, immunofluorescence and Western blotting were used to explore the downstream signaling pathway involved in the ALYREF-XRCC6 axis. Results: In this study, ALYREF was found to be highly expressed in EC and correlated with poor prognosis. Elevated expression of ALYREF was shown to promote EC progression. Mechanistically, XRCC6 was identified as a downstream target transcript of ALYREF. ALYREF regulated the stability of XRCC6 mRNA through an m5C-dependent mechanism. The ALYREF-m5C-XRCC6 axis promoted EC progression by activating the Wnt/β-catenin signaling pathway. Conclusions: Our findings indicate that ALYREF functions as an oncogenic factor in EC. Moreover, the ALYREF-m5C-XRCC6 axis drives EC progression and represents a potential therapeutic target.
