Hakai links m6A RNA methylation to immune regulation in colorectal cancer

BackgroundThe mA modification is the most abundant RNA epigenetic mark in colorectal cancer (CRC). In this study, we focused on Hakai, a methyltransferase writer-associated protein whose molecular function within the complex and link to CRC immune regulation remain poorly characterized. MethodsWe performed RNA-seq to assess transcriptomic changes after Hakai silencing in CRC models and MeRIP-seq to profile methylation alterations. Validation was carried out by Western blot, flow cytometry, ELISA, and RT-qPCR. Protein interactions within the writer complex were analysed by co-immunoprecipitation, and changes in protein localization by cell fractionation assays. Peripheral blood from healthy donors and lamina propria mononuclear cells from CRC patients were incubated with supernatants from Hakai-silenced CRC cells to evaluate effects on immune responses. ResultsHakai silencing induced phenotypic changes in both monolayer and 3D CRC cultures, while its impact on the global transcriptome was limited. However, significant alterations in the methylation of immune response-related genes and reduced total mA levels were observed. Hakai interacted with the writer-associated protein VIRMA, and its silencing altered the subcellular localization of METTL3. Moreover, conditioned media from Hakai-silenced CRC cells modulated the expression of immune response markers in patient-derived gut immune cells. ConclusionsHakai acts as a component of the mA writer complex in CRC cells, influencing RNA methylation and the expression of immune-related genes. These findings suggest that Hakai silencing may enhance antitumour immune activity and represent a potential strategy to boost cancer immunity in colorectal cancer.