Abstract
BACKGROUND: Glioblastoma (GBM), the most aggressive primary brain tumor, has a dismal prognosis largely due to therapy-resistant stem-like cells that drive recurrence. While N6-methyladenosine modifications in GBM are well-studied, the role of 5-methylcytosine (m(5)C) modifications specifically in circular RNAs (circRNAs) remains poorly understood. METHODS: Bioinformatic analysis, qRT-PCR and Western blot assays were used to investigate the expression of NSUN7, circNTRK2, YBX3, STK31, IKZF1 in GBM tissues and cell lines. m(5)C dot blot assays, m(5)C-bisulfite sequence assays, Sanger’s sequencing, RNA pull down and RIP assays, in vitro kinase assays, chromatin immunoprecipitation and luciferase reporter gene assays are used to clarify the interaction between factors above. Colony formation assays, sphere formation assays, and stemness marker (OCT4, DCLK1) analysis were utilized to assess the impact of the factors above on GBM stemness. Subcutaneous heterotopic and orthotopic xenograft are utilized to demonstrate the function of YBX3/circNTRK2/STK31/IKZF1 axis in GBM in vivo. RESULTS: NSUN7 and YBX3 are both upregulated in GBM tissues and cell lines. NSUN7 catalyzes m(5)C modification of circNTRK2. YBX3 decreasing circNTRK2 stability and function via binding m(5)C of circNTRK2. CircNTRK2 binds to and activates STK31, leading to phosphorylation of IKZF1 at S(63), which decreases the O-GlcNAc levels and transcriptional activity of IKZF1, thereby promoting the stem cell characteristics of GBM cells. CONCLUSIONS: This study unveils that NSUN7-mediated m(5)C modification of circNTRK2 fuels GBM stemness via circNTRK2-dependent STK31 activation, thus identifying potential biomarkers for targeted molecular therapy of GBM and providing novel therapeutic targets for GBM treatment. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12967-025-07484-1.