STUB1-mediated K63-linked ubiquitination of UHRF1 promotes the progression of cholangiocarcinoma by maintaining DNA hypermethylation of PLA2G2A

Cholangiocarcinoma (CCA) is a highly malignant tumor characterized by a lack of effective targeted therapeutic strategies. The protein UHRF1 plays a pivotal role in the preservation of DNA methylation and works synergistically with DNMT1. Posttranscriptional modifications (PTMs), such as ubiquitination, play indispensable roles in facilitating this process. Nevertheless, the specific PTMs that regulate UHRF1 in CCA remain unidentified. We confirmed the interaction between STUB1 and UHRF1 through mass spectrometry analysis. Furthermore, we investigated the underlying mechanisms of the STUB1-UHRF1/DNMT1 axis via co-IP experiments, denaturing IP ubiquitination experiments, nuclear‒cytoplasmic separation and immunofluorescence experiments. STUB1-UHRF1/DNMT1-mediated DNA methylation plays a crucial role in promoting the epigenetic silencing of tumor suppressor genes (TSGs) and facilitating tumor progression. To investigate the specific TSGs regulated by the STUB1-UHRF1/DNMT1 axis in CCA cells, RNA-seq analysis of overexpressed STUB1 and negative control TFK1 cells was performed. To investigate the specific tumor suppressor genes (TSGs) regulated by the STUB1-UHRF1/DNMT1 axis in CCA cells, RNA-seq analysis of overexpressed STUB1 and negative control TFK1 cells (with 3 replicates in each group) was performed.