Abstract: Objective To explore the role of TRIM21 in modulating the invasive phenotype of hepatocellular carcinoma (HCC) cells and its mechanism of action.Methods RNA interference technique was used to knock down the expression of TRIM21 and β-catenin, alone or in combination, in HCC cell lines 97H and LM3, and the interfering efficiency and the activity of closely related pathways were determined using Western blotting. The two cells with TRIM21 knockdown (siTRIM21 97H and siTRIM21 LM3 cells) were assessed for their invasion ability in vitro using Transwell invasion assay, and the lung metastasis capacity of siTRIM21 LM3 cells following tail vein injection was evaluated in nude mice. The binding of TRIM21 with β-catenin and the ubiquitylation level of β-catenin in TRIM21-overexpressing HEK293 cells were determined with Western blotting and co-immunoprecipitation assay. We also compared the overall survival of patients with CTNNB1highTRIM21high and CTNNB1highTRIM21low HCC subtypes using Kaplan-Meier method based on filtrated and grouped HCC clinical data from TCGA database.Results TRIM21 knockdown significantly enhanced the invasion ability of 97H and LM3 cells in vitro (P<0.01 or 0.05) and the lung metastasis ability of LM3 cells in nude mice (P<0.01), and simultaneous knockdown of β -catenin obviously suppressed the in vitro invasiveness of the cells (P<0.0001 or 0.05). Co-immunoprecipitation assay showed that TRIM21 was capable of directly binding with β-catenin protein to accelerate the ubiquitination and degradation of the latter, leading to inhibition of nuclear translocation of β-catenin and hence reduced invasiveness of HCC cells. Bioinformatic analysis showed that compared patients with CTNNB1highTRIM21low HCC subtype where Wnt pathway was activated, the patients with CTNNB1highTRIM21high HCC subtype had a significantly better survival outcomes (P<0.05).Conclusion A high expression of TRIM21 suppresses the invasion of HCC cells by promoting β-catenin ubiquitylation and degradation, which possibly explains the poor prognosis of CTNNB1highTRIM21low HCC patients.

Key words: liver cancer; aggressiveness; TRIM21; β-catenin; ubiquitination; prognosis