HIF-1α-induced Expression Of M6A Reader YTHDF1 Drives Hypoxia-induced Autophagy And Malignancy Of Hepatocellular Carcinoma

Background&Aims:Hepatocellular carcinoma（HCC）is one of the most prevalent,lethal tumors,with China accounting for 50%of the global cases.Despite the tremendous progress in HCC treatment,the treatment options remain limited.Hence,new therapeutic strategies are needed to address these limitations.Hypoxia resulting from rapid growth,accompanied by insufficient blood supply causes microenvironment stress in solid tumors.Previous studies have demonstrated that autophagy is a crucial adaptive feature that favors tumor survival under hypoxic conditions by clearing damaged organelles,regulating production of reactive oxygen species,and decreasing apoptosis.Thus,suppression of autophagy is a promising strategy for developing antitumor therapies.Methylation modification resulting in N6-methyladenosine（m6A）has been recognized as the most prevalent internal modification,ubiquitously occurring in the eukaryotic m RNAs.The m6A modification can influence m RNA decay,splicing,transport,localization,and translation.Accumulating evidence has demonstrated that the m6A modification is associated with various biological processes,including stem cell differentiation,tissue development,and tumor progression.A recent study showed that tumor hypoxia results in m6A epigenetic remodeling.Hypoxia-inducible factors（HIFs）play a vital role in m6A modification.However,the biological significance of m6A under hypoxia and the underlying regulatory mechanisms,especially in hypoxia-induced autophagy in human HCC,remain elusive.Methods:The prognostic value of YTHDF1 expression was evaluated using the tissue microarray（TMA）and its clinicopathological characteristics.Autophagy was detected using m RFP-GFP-LC3 immunofluorescence staining,western blotting,and transmission electron microscopy（TEM）.Chromatin immunoprecipitation（Ch IP）,RNA Me RIP-sequencing,proteomics,RNA immunoprecipitation（RIP）,and polysome profiling were used to explore both upstream and downstream regions of YTHDF1 in HCC.Multiple HCC models including HCC cells,HCC organoids,YTHDF1^hep-/-mice,and nude mice were employed to confirm the effects of YTHDF1 expression alteration on HCC hypoxia-induced autophagy and autophagy-related malignancy.Results:A significant correlation between YTHDF1 and hypoxia-induced autophagy was observed in HCC cell lines.Significant overexpression of YTHDF1 was observed in the HCC tissues associated with poor prognosis.HIF-1αregulated YTHDF1transcription by directly binding to its promoter region under hypoxia.Multiple HCC models including HCC cells,HCC organoids,YTHDF1^hep-/-mice,and nude mice were employed to confirm that YTHDF1 deficiency inhibits HCC hypoxia-induced autophagy and autophagy-related malignancy.YTHDF1 contributes to the translation of autophagy-related genes,ATG2A and ATG14,in an m6A-dependent manner,thus facilitating autophagy and autophagy-related tumorigenesis and metastasis of HCC.Conclusion:HIF-1α-induced YTHDF1 expression was associated with hypoxia-induced autophagy and autophagy-related HCC progression via promoting the translation of autophagy-related genes ATG2A and ATG14 in a m6A-dependent manner,suggesting that YTHDF1 is a potential prognostic biomarker and therapeutic target for patients with HCC.