RNF216 Drives Hepatocellular Carcinoma Progression by Ubiquitin‐Dependent Degradation of the Tumour Suppressor CYLD

BACKGROUND AND AIMS: The role of ubiquitination in the progression of hepatocellular carcinoma (HCC) remains unclear. This study investigated ubiquitination heterogeneity across cell types and the function of the E3 ubiquitin ligase RNF216 in HCC. METHODS: To map the cellular landscape and ubiquitination heterogeneity, we performed single-cell RNA sequencing (scRNA-seq) on 10 HCC samples. Bioinformatics analysis identified RNF216 as a key prognostic ubiquitination-related gene (URG), validated in public datasets and an internal cohort by immunohistochemistry. Functional assays (CCK-8, wound healing, and Transwell migration and invasion) and a xenograft model were performed in HCC cells. The mechanisms were explored using Western blotting, co-immunoprecipitation, ubiquitination assays, and immunofluorescence. RESULTS: ScRNA-seq revealed the highest ubiquitination activity in T and B cells and the lowest in hepatocytes. Differential expression analysis between high- and low-ubiquitination groups identified 75 URGs. Among these, RNF216 was significantly upregulated in HCC and correlated with advanced stage and unfavourable prognosis, prompting its selection for further functional validation. RNF216 knockdown suppressed proliferation, migration, invasion, and EMT in both Huh7 and MHCC97H cells, and inhibited tumour growth in vivo. Mechanistically, RNF216 directly interacted with CYLD and promoted its ubiquitination and proteasomal degradation. CYLD re-expression in RNF216-overexpressing Huh7 cells significantly reversed RNF216-induced increases in proliferation, migration, invasion, and EMT. Immunofluorescence demonstrated that RNF216 promotes β-catenin nuclear translocation in a CYLD-dependent manner. CONCLUSIONS: RNF216 promotes HCC progression through ubiquitination and degradation of CYLD, leading to β-catenin accumulation and EMT. RNF216 may serve as a therapeutic target for HCC.