The Role Of Deubiquitinating Enzyme USP16 Expression Down-regulation In The Effect Of Hepatitis B Virus X Protein On Hepatocarcinogenesis

Background and Objective : Hepatitis B virus(HBV)infection has been recognized as the major event that contributes to the development of hepatocarcinoma(HCC).HBV X protein(HBx)is an oncogenic factor that accelerates HCC progression by promoting tumor growth and metastasis.Mounting evidence has demonstrated that COOH-terminal truncated HBx is frequently detected in clinical HBV-related HCC.Deubiquitinases(DUBs)are critical for various cellular processes,including cell proliferation,differentiation and apoptosis,through regulating protein stability and activity.The dysregulation of DUBs has been associated with the development of many diseases,including cancers.In this study,we report that COOH-terminal truncated HBx can negatively regulate USP16 expression.The aim of this study is to explore whether and how USP16 functions in regulating the biological properties of liver tumor cells,and to address the relevance of HBx-mediated downregulation of USP16 in the development of HCC.Methods: The LO2 and HepG2 cells stably expressing the COOH-terminal truncated HBx proteins(HBx△35 and HBx△14)were generated for identifying the DUBs that could be transcriptionally controled by COOH-terminal truncated HBx.A total 40 DUBs were screened for measuring the alterations of their mRNA levels in cells with or without the expression of HBx△35 or HBx△14 by Real-time RT-PCR.The mRNA expression of USP16 and the status of HBx were examined in 26 paired HCC samples and their adjacent non-cancerous liver tissues.Liver tumor cells were lentivirally transduced with USP16 or the sh RNAs targeting USP16.Cell proliferation,cell cycle distribution,anoikis-resistance and in vivo tumor growth abilities were analyzed by MTT,clone formation,flow cytometry and xenograft tumor formation.The stem-cell properties of the liver tumor cells as well as their resistance to chemo-reagents were also examined.The downstream events of USP16 were delineated through microarray and IPA analysis;the changes of gene expression revealed by array analysis were verified by RT-PCR.Finally,the expression pattern of USP16 in 100 paired HCC samples was examined and its clinical relevance was analyzed.Results: Ectopic expression of the COOH-terminal truncated HBx proteins,HBx△35 and HBx△14,resulted in a consistent downregulation of USP16 expression in LO2,HepG2,Huh7 and PLC/PRF/5 cells.The treatment of the NF-κB inhibitor BAY11-7082 could alleviate the downregulation of USP16 expression in cells with HBx△35 expression.We further examined the levels of USP16 transcripts in 26 paired HCC tumor samples,and found that the mRNA abundancy of USP16 in the non-tumor tissues with the presence of the full-length HBx was significantly lower than that in the tumor tissues positive for COOH-terminal truncation of HBx.Knockdown of USP16 significantly promoted cell proliferation and inhibited anoikis in Huh7 and PLC/PRF/5 cells.Furthermore,USP16 knockdown efficiently increased the tumor growth of Huh7 cells.In sharp contrast,ectopic expression of USP16 abrogated the promoting activities of HBx△35 on cell proliferation,tumor growth and anoikis-resistance.In addition,knockdown of USP16 significantly increased spheroid-forming capability,mRNA expression of the stemness-related genes,such as Sox2,Nanog,CD44 and CD133,as well as the resistance to 5-Fu and adriamycin in Huh7 and PLC/PRF/5 cells.Consistently,USP16 overexpression counteracted HBx△35-mediated enhancement of chemo-resistance and stem-cell properties in these tumor cells.Through genome transcriptional analysis,we found that the levels of the PTEN signaling-related genes,including GSK3β,KRAS,FLT4,ITGA2 and BCL2,were regulated by USP16 knockdown.Importantly,USP16 silencing significantly decreased protein levels of PTEN in tumor cells.Finally,we measured USP16 protein levels in 100 paired human HCC samples,and found that USP16 expression was downregulated in tumor tissues and inversely correlated with clinical stage.Conclusions:COOH-terminal truncated HBx negatively regulates USP16 expression in liver tumor cells.USP16 inhibits liver tumor cell growth,sensitizes cells to anoikisinduction and chemo-drug treatment,and decreases stem-cell properties.USP16 is capable of counteracting the protumorigenic activities of COOH-terminal truncated HBx.USP16 modulates PTEN signaling and other key pathways in liver tumor cells.USP16 expression is downregulated in tumor tissues and inversely correlates with clinical stage.Therefore,our results indicate that USP16 downregulation by COOHterminal truncated HBx may contribute to HCC development and progression.