| Hepatocellular carcinoma(HCC)is malignant tumour that occurs in the liver tissue and metastasis recurrence is the main reason for the high mortality rate of HCC.Hepatitis B virus(HBV)infection is an important cause of the high incidence of liver cancer in China.Death Associated Protein Kinase 1(DAPK)is a serine/threonine protein kinase that can be regulated by interferon-γ(IFN-γ).The expression of DAPK is frequently downregulated in many human tumor tissues.Early studies have demonstrated significantly lower levels of DAPK expression in hepatocellular carcinoma tissues compared to paraneoplastic tissues,and low DAPK expression has been identified as an unfavorable prognostic factor for patients with hepatocellular carcinoma.Further mechanistic studies on DAPK in hepatocellular carcinoma have shown that DAPK can inhibited the CDC42-integrin pathway through DDX20 to achieve their anti-cancer function,and that DAPK can enhance the protein stability of DDX20 and upregulate DDX20 protein expression through the proteasome pathway without affecting DDX20 m RNA levels.Therefore,to further explore how DAPK regulates DDX20 expression through the proteasome pathway becomes the core of this research.The ubiquitin-proteasome degradation pathway is the main pathway for protein degradation in eukaryotic cells.In this study,the ubiquitination modification of DDX20 was detected by drug experiments with MG132 and immunoprecipitation of ubiquitinated proteins,which showed that there were multiple ubiquitination modifications of DDX20,mainly K11 and K48 modifications.The ubiquitination modification of substrate proteins by ubiquitin ligase E3 is a determining factor for the specific recognition of target proteins during ubiquitin-proteasome degradation.The protein profile revealed that TRIM25(Tripartite Motif Containing 25)interacted with DDX20,which was further confirmed by immunoprecipitation and fluorescence co-localization experiments.WB assay showed that the expression of both endogenous and exogenous DDX20 was negatively regulated by TRIM25,while the protein half-life assay and ubiquitination assay showed that TRIM25 reduced the stability of DDX20 protein and increased the ubiquitination of DDX20.To further explore how DAPK increases DDX20 protein expression through the ubiquitin-proteasome pathway,we examined the effect of DAPK on the ubiquitination level of DDX20.The results showed that overexpression of DAPK significantly downregulated the ubiquitination level of internal and external DDX20.Fluorescence co-localization assays and immunoprecipitation results showed that DAPK and DDX20 can form protein complexes in cells.Ubiquitination assays and truncation assays revealed that the 1-364 aa region of DAPK binds intracellularly to the DDX20 1-244 aa region,which is a key region in the regulation of DDX20 ubiquitination and protein expression by DAPK.However,in vitro protein interaction assays showed that DAPK 1-364 did not bind directly to the DDX20 1-244 region,indicating the involvement of an intermediate protein in between them.Subsequent protein immunoprecipitation experiments demonstrated intracellular interaction between DAPK and TRIM25.Co-expression of DAPK,DDX20,and TRIM25 resulted in formation of an intracellular protein complex.When TRIM25 was knocked down in cells co-expressing DAPK and DDX20,immunoprecipitation results showed that knockdown of TRIM25 affected the intracellular binding of DAPK to DDX20,indicating that TRIM25 plays a role in DAPK regulation of DDX20.The GEO database and cell experiments demonstrated that HBV/HBx upregulates the expression of DAPK,significantly enhances the expression of DDX20 protein,prolongs its half-life,and inhibits its ubiquitination.The GEO database and cell experiments demonstrated that HBV/HBx upregulates the expression of DAPK,significantly enhances the expression of DDX20 protein,prolongs its half-life,and inhibits its ubiquitination.Meanwhile,RNA-seq data were used to construct HBV-induced dysregulated coding genes and non-coding gene networks.By integrating RNA-seq data from HBV-infected cells with that of DDX20-knockdown hepatoma cells,it was discovered that DDX20 plays a crucial role in the process of HBV infection by regulating transcription factors,integrin binding,NF-κB signaling,mi RNA processing and other physiological processes.In conclusion,the findings of this study can be briefly summarized as follows:1)DDX20 is degraded via the ubiquitin-proteasome pathway;TRIM25 is the E3 ubiquitin ligase of DDX20.2)DAPK improves DDX20 protein stability by inhibiting the ubiquitin-proteasome pathway of DDX20,in which DAPK does not bind directly to DDX20.Knockdown of TRIM25 can affect the intracellular binding of DAPK to DDX20.DAPK,TRIM25,and DDX20 form a protein complex in the cell.3)HBV/HBx can activate DDX20 through DAPK,reduce DDX20 ubiquitination,improve protein stability and enhance its protein expression.4)DDX20 affects various pathways such as transcriptional regulation,integrin binding in HBV related HCC.Currently,the DAPK-TRIM25-DDX20 pathway has not been reported.In the present study,the E3 ligase that regulates DDX20 was discovered,which is rather innovative.In addition,the molecular mechanism of DAPK regulating DDX20 in pre-existing hepatocellular carcinoma was explored in more insight,the activation of the DAPK-TRIM25-DDX20 pathway after HBV infection has been discovered,and the possible signal pathways regulated by DDX20 after HBV infection were screened.All these studies will help to further understand the molecular mechanism of HBV related HCC and lay the theoretical foundation for the subsequent search for HBV related HCC markers,the construction of predictive models and the exploration of more effective therapeutic approaches. |
