YTHDF2 Promotes Hepatocellular Carcinoma Progression By Regulating ETV5-PD-L1/VEGFA Axis

Objective:Hepatocellular carcinoma（HCC）is associated with high malignancy rates.N6-methyladenosine（m⁶A）modification,as the most abundant RNA modification,widely participates in multiple diseases progression,especially in cancer development.Recently,YTH N6-methyladenosine RNA binding protein 2（YTHDF2）has been revealed to play vital roles in various malignancies,but its role and function are not fully understood in hepatocellular carcinoma（HCC）.Methods:The association of YTHDF2 expression with the clinicopathological characteristics and prognosis in patients with HCC was assessed by quantitative real-time PCR（RT-q PCR）,western blot,immunohistochemistry and public datasets.In vitro and vivo function experiments（T cell-killing assay,tube formation assay,DEN/CCl₄-induced hepatocarcinogenesis,orthotopic liver tumor model,and so on）were conducted to investigate the role of YTHDF2 in HCC.High-throughput sequencing was performed to screen the downstream targets of YTHDF2.Furthermore,RNA immunoprecipitation,RNA pulldown,GST pulldown,RT-q PCR,western blot,luciferase reporter assays and chromatin immunoprecipitation assays were further applied to dissect the detailed mechanisms.Subsequently,the orthotopic liver tumor model was performed,and the treatment of intrapitoneal injection of Programmed death ligand-1（PD-L1）monoclonal antibody plus anti-angiogenesis therapy,and siRNA-YTHDF2 in the aptamer/liposome was injected into the tail vein to explore the significance of YTHDF2 in the treatment of HCC.Results:Clinical investigation showed that YTHDF2 is frequently upregulated in HCC,and the upregulated YTHDF2 in HCC predicts a worse prognosis.YTHDF2 promotes the immune evasion and angiogenesis by enhancing the expression of PD-L1 and Vascular endothelial growth factor A（VEGFA）,and subsequently facilitating HCC progression.Mechanistically,YTHDF2 promotes the translation of ETS variant transcription factor 5（ETV5）mRNA in an m⁶A-dependent manner,resulting in transcriptional activation of PD-L1 and VEGFA.More specifically,YTHDF2 interacts with Eukaryotic translation initiation factor 3 subunit B（eIF3b）directly,thereby facilitating the translation of ETV5.Consistently,the treatment combined PD-L1 antibodies and anti-angiogenic therapy targets YTHDF2-overexpression HCC cells preferentially.Besides,suppression of YTHDF2 by the siRNA-containing aptamer/liposomes revealed an antitumor effect in HCC.Conclusions:We revealed that YTHDF2 increased PD-L1 and VEGFA expression by facilitating the translation of ETV5 in HCC.And we proposed YTHDF2 as a potential prognostic marker and therapeutic targets for HCC.