Abstract
Background: Hepatocellular carcinoma (HCC) maintains a significant mortality burden, primarily attributable to the lack of validated prognostic molecular signatures. While N6-methyladenosine (m6A) reader proteins, especially YT521-B homology domain family proteins (YTHDFs), show emerging relevance in oncogenesis, their regulatory interplay and clinical utility as combinatorial biomarkers remain unclear. Our study aimed to explore the regulatory network and clinical relevance of YTHDF paralogs in HCC. Methods: Spatial co-expression patterns were analyzed via immunohistochemistry and spatial transcriptomics in HCC tissues. Protein/RNA levels of YTHDFs were quantified by western blot and quantitative real-time polymerase chain reaction (PCR). RNA immunoprecipitation (RIP), enhanced crosslinking and immunoprecipitation (e-CLIP) sequencing, dual fluorescence reporter assay, and RNA stability experiments were conducted to verify underlying regulatory mechanisms. Univariate and multivariate Cox regression analysis and Kaplan-Meier survival analysis were utilized to estimate the potential clinical significances. Results: YTHDF1 overexpression correlated with poor prognosis and emerged as the strongest prognostic marker among the YTHDF paralogs. YTHDF1 could bind to the messenger RNA (mRNA) of YTHDF2 and YTHDF3 at specific m6A sites. However, in vitro and in vivo experiments revealed that YTHDF1 did not impact the mRNA and protein levels of YTHDF3, and instead enhanced the mRNA stability and translation efficiency of YTHDF2 in an m6A-dependent manner. Interestingly, YTHDF1 and YTHDF2 exhibited comparable spatial expression patterns and demonstrated correlation in terms of spatial features and expression levels in HCC, which had not been previously reported. Clinical analysis conducted on two clinical cohorts corroborated the notable correlations between YTHDF1 and YTHDF2, and revealed that HCC patients with higher expression of both proteins had worse prognosis. Conclusions: YTHDF1 drives HCC progression through m6A-mediated stabilization of YTHDF2, revealing their functional synergy. Their combined spatial and expression profiles offer a superior prognostic biomarker, suggesting novel therapeutic targets.