a PLC-8024 and MIHA cells were transiently transfected with Vec or RALYL plasmid for 48 h and then treated with ActD for 0, 2, 4, 6, 8, 10 h. The TGF-β2 mRNA level was determined using qRT-PCR and normalized to 18S rRNA. The GAPDH mRNA level was used as a negative control. The values represent the mean ± SD of 3 independent experiments (*P < 0.05, **P < 0.01, independent Student’s t-test). b RIP assay using total cell lysates of Huh7 cells was used to assess the interaction between RALYL and TGF-β2 mRNA. Enrichment of TGF-β2 mRNA in the RALYL-containing immunoprecipitated particles was measured using qRT-PCR and normalized to input. c m6A RIP and qRT-PCR were used to determine the percentage of TGF-β2 mRNA with m6A modification in 8024-Vec and 8024-RALYL cells (*P < 0.05, Student t-test). d 3′UTR of TGF-β2 mRNA was fused with firefly luciferase reporter. RALYL overexpression augmented the luciferase activity in 8024 and MIHA cells. The values represent the mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, two-sided Student’s t-test). e The interaction of RALYL with FTO was determined by co-immunoprecipitation with anti-FTO (IP: FTO) and anti-FLAG antibody (IP: FLAG) or IgG (IP: IgG) in RALYL-flag-transfected cells. Total cell lysate (Input) was used as a positive control. f A schematic diagram illustrating the proposed RALYL enhanced stemness-related features in HCC. RALYL could decrease m6A modification of TGF-β2 mRNA, thereby maintain its stability by interaction with FTO. By sustaining the secretion of TGF-β2, the PI3K/AKT and STAT3 were activated to increase the stemness of HCC.