METTL4 enhances GLI1 translation through m6Am modification to promote tumor progression as a therapeutic target for hepatocellular carcinoma

• First identification of METTL4-mediated m 6 Am modification on GLI1 mRNA as a driver of Hedgehog pathway hyperactivation in HCC. • Multi-cohort validation (n = 802) establishes METTL4 as an independent prognostic marker across HBV/HCV/NAFLD-related HCC subtypes. • Mechanistic dissection reveals METTL4 exclusively regulates translation efficiency rather than stability of oncogenic transcripts via 5′UTR-proximal m6Am deposition. • Clinically translatable siRNA-nanoparticle achieves >70 % tumor growth suppression with undetectable organ toxicity in spontaneous HCC models. Hepatocellular carcinoma (HCC) is a major cause of cancer-related mortality. Epigenetic dysregulation, particularly through methyltransferase METTL4, plays a critical role in HCC progression. METTL4 is known to catalyze m 6 Am methylation of mRNA, yet its pathological significance in liver cancer remains unclear. This study aims to firstly investigate the role of METTL4 in the pathogenesis of HCC, and then to evaluate the antitumor efficacy of targeting METTL4 in inhibiting HCC growth and metastasis. In a total of 802 postoperative HCC patients from four independent cohorts, we analyzed METTL4 expression in HCC tissues and its correlation with tumor staging and survival rates. Functional assays assessed the impact of METTL4 knockdown on HCC cell proliferation and metastasis, and patient-derived organoids (PDO) were used to evaluate the therapeutic efficacy of targeting METTL4. m 6 A-seq analyses identified GLI1 as a direct target of METTL4, investigating how METTL4-mediated m 6 Am modification affects GLI1 mRNA translation. Finally, we developed a siMETTL4-carrying nanoparticle and explored their efficacy in treating HCC in orthotopic and spontaneous liver tumor models. METTL4 was significantly upregulated in HCC tissues, correlating with higher tumor staging and poorer survival. Knockdown of METTL4 significantly inhibited HCC cell proliferation, migration, tumor growth, and lung metastasis. Meanwhile, targeting METTL4 effectively inhibited the growth of HCC PDO. METTL4-mediated m 6 Am modification enhanced GLI1 mRNA translation, with recognized target sites confirmed. Finally, siMETTL4-encapsulated nanoparticles effectively inhibited the growth and metastasis of orthotopic and spontaneous liver tumors. Our findings highlight the role of METTL4-mediated m 6 Am modification in promoting HCC progression by enhancing GLI1 translation. Targeting METTL4 with siMETTL4-Nanoparticles represents a novel therapeutic strategy, bridging molecular insights in RNA epitranscriptomics with translational applications for HCC treatment.