TRIM21可抑制肝癌细胞的侵袭能力:基于泛素化途径降解β-catenin

TRIM21 suppresses invasion of hepatocellular carcinoma cells by promotingβ-catenin ubiquitylation and degradation

目的探讨TRIM21基因在肝癌侵袭表型中的作用及机制。方法利用siRNA敲低肝癌细胞MHCC 97H,HCC LM3中的TRIM21、β-catenin基因,将肝癌细胞分为对照组:转染对照siRNA;TRIM21敲低组:转染siTRIM21 RNA;CTNNB1敲低组:转染siCTNNB1 RNA;双敲低组:共转染siTRIM21 RNA及siCTNNB1 RNA。利用Western blotting验证敲除效率及相关通路的激活或抑制情况。通过Transwell侵袭实验检测肿瘤细胞的侵袭性。通过尾静脉注射敲低TRIM21的HCC LM3细胞构建裸鼠肺转移模型以观测其肺转移能力。在HEK 293细胞中过表达TRIM21,将HEK 293细胞分为对照组:加入转染试剂;过表达组:转染flagTRIM21。并利用Co-IP检测TRIM21蛋白与β-catenin的相互作用及泛素化水平。生信分析TCGA数据库中CTNNB1^(high)TRIM21^(high)和CTNNB1^(high)TRIM21^(low)两种亚型肝癌的预后。结果敲低TRIM21可以显著增强肝癌细胞MHCC 97H和HCC LM3的侵袭能力(P<0.01,P<0.05)和HCC LM3细胞的肺转移能力(P<0.01)。机制研究发现,TRIM21可通过泛素化蛋白酶体途径降解β-catenin以减少其入核,进而降低肝癌细胞的的侵袭性(P<0.0001,P<0.05)。由于TRIM21表达水平降低使肝癌细胞中Wnt信号通路激活,最终使CTNNB1^(high)TRIM21^(high)亚型肝癌患者的生存预后明显优于CTNNB1^(high)TRIM21^(low)亚型肝癌患者(P<0.05)。结论TRIM21能通过泛素化途径降解β-catenin来抑制肝癌细胞的侵袭性,这可能导致了CTNNB1^(high)TRIM21^(low)肝癌患者预后较差。

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 andβ-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(siTRIM2197H 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 withβ-catenin and the ubiquitylation level ofβ-catenin in TRIM21-overexpressing HEK293 cells were determined with Western blotting and co-immunoprecipitation assay.We also compared the overall survival of patients with CTNNB1^(high)TRIM21^(high) and CTNNB1^(high)TRIM21^(low) 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 ofβ-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 withβ-catenin protein to accelerate the ubiquitination and degradation of the latter,leading to inhibition of nuclear translocation ofβ-catenin and hence reduced invasiveness of HCC cells.Bioinformatic analysis showed that compared patients with CTNNB1^(high)TRIM21^(low) HCC subtype where Wnt pathway was activated,the patients with CTNNB1^(high)TRIM21^(high) HCC subtype had a significantly better survival outcomes(P<0.05).Conclusion A high expression of TRIM21 suppresses the invasion of HCC cells by promotingβ-catenin ubiquitylation and degradation,which possibly explains the poor prognosis of CTNNB1^(high)TRIM21^(low) HCC patients.