To investigate how deubiquitinase OTU domain-containing protein 3 (OTUD3) suppresses the progression of hepatocellular carcinoma via gut-liver axis metabolic remodeling and microbiome dynamics.

A total of 24 male 2-week-old littermate C57BL/6J mice (12 wild-type and 12 Otud3^-/-) were divided into two differential genotype groups before 6 mice from each group were randomly chosen to receive intraperitoneal injections of N-nitrosodiethylamine (DEN) for hepatocellular carcinoma (HCC) induction. The mice were divided into four groups (n=6/group): Otud3^+/+ control (WT CON), Otud3^-/- control (KO CON), Otud3^+/+ DEN-induced HCC (WT DEN), and Otud3^-/- DEN-induced HCC (KO DEN). At 40 weeks of age, liver tissues were collected for metabolomic profiling, and fecal samples were obtained for 16S rRNA sequencing.

Multivariate analyses, including principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA), sparse partial least squares-discriminant analysis (sPLS-DA), and orthogonal partial least squares-discriminant analysis (OrthoPLS-DA), demonstrated complete intergroup separability. Fifty-four differential metabolites were identified between the WT DEN and KO DEN groups through metabolomic profiling, with gut-liver axis-associated pathways such as cholesterol metabolism and fatty acid biosynthesis revealed by KEGG pathway analysis. Microbiome analysis indicated an upregulation of Bacteroides at the genus level in the KO DEN group compared to WT DEN. Pearson correlation analysis highlighted amino acids and derivatives as predominant metabolite classes and revealed Bacteroidetes and Firmicutesas the dominant gut microbial phyla.

OTUD3 suppresses HCC progression by modulating gut-liver axis metabolism, potentially mediated by elevated betaine and increased abundance of Odoribacter, Alistipes, and Lachnoclostridium.