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Background Radiation‐induced hepatic stellate cell (HSC) activation promotes radiation‐induced liver fibrosis (RILF), a complication for hepatocellular carcinoma (HCC) radiotherapy. The demethylase alpha‐ketoglutarate‐dependent dioxygenase alkB homolog 5 (ALKBH5) decreases N6‐methyladenylate methylation (m 6 A) modification of RNA, while its role in regulating RILF pathogenesis and HCC radiosensitivity remains unknown. Methods Methylated RNA immunoprecipitation sequencing (MeRIP‐seq) and RNA‐sequencing (RNA‐seq) were used to screen target genes regulated by ALKBH5. HSC with altered ALKBH5 expression was used to assess irradiation‐induced HSC activation and the effect of HSC on recruitment and polarisation of monocytes. Key cytokines in medium from irradiated HSC‐educated monocytes were identified by cytokine array detection. The effects of blocking ALKBH5 and key cytokines on RILF and HCC radiosensitivity were also evaluated. Results Radiation‐induced ALKBH5 expression in HSC mediated m 6 A demethylation of toll‐interleukin 1 receptor domain containing adaptor protein (TIRAP) mRNA and activated its downstream NF‐κB and JNK/Smad2 pathways to promote HSC activation. Additionally, ALKBH5 regulated CCL5 secretion by irradiated HSC to promote monocyte recruitment and M2 macrophage polarisation. Notably, polarised monocytes secreted CCL20 to up‐regulate ALKBH5 expression in HSC, and reduce HCC radiosensitivity by activating ALKBH5/TIRAP axis in HCC cells. ALKBH5 knockdown‐combined CCR6 (CCL20 receptor) inhibitor significantly alleviated RILF and improved HCC radiosensitivity in mice. HCC patients with high ALKBH5 and TIRAP expression were prone to radiation‐induced liver injury and poor tumour response to radiotherapy. Conclusions Collectively, irradiation up‐regulates ALKBH5 in HSC to mediate monocyte recruitment and M2 polarisation and form positive feedback to promote RILF and reduce HCC radiosensitivity. The dual roles of ALKBH5 as a microenvironmental regulator and radiosensitisation target provide new ideas for RILF prevention and radiosensitisation of HCC.

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