RESUMO
Objective:To investigate the effects of the interaction between ubiquitin-specific peptidase 22 (USP22) and hepatitis B virus X protein (HBx) on the protein level and the biological function of HBx.Methods:The interactions between HBx and USP22 were analyzed by GST pull-down, co-immunoprecipitation assay and confocal laser scanning assay. USP22 recombinant plasmids or specific siRNA were transiently co-transfected with HBx plasmids. Western blot were used to detect the protein level of HBx. The half-life and degradation pathway of HBx in the transfected cells treated with cycloheximide (CHX) or proteasome inhibitor MG132 were detected. In vivo ubiquitination assay was used to detect the ubiquitination of HBx with USP22 overexpression. Moreover, dual-luciferase reporter assay and colony formation assay were used to analyze the effects of USP22 on the biological function of HBx. Results:USP22 could interact with HBx in vivo and in vitro. USP22 significantly increased the stability of HBx and inhibited the proteasome-mediated degradation of HBx protein by reducing the ubiquitination of HBx, thereby enhancing the biological function of HBx. Conclusions:USP22 inhibited HBx protein degradation through ubiquitin-dependent proteasome pathway, thus enhancing the stability and biological function of HBx.
RESUMO
Objective: To investigate the effect of microRNA-34b (miR-34b) on the proliferation of bladder cancer BIU-87 cells, and to exploreObjective: To investigate the effect of microRNA-34b (miR-34b) on the proliferation of bladder cancer BIU-87 cells, and to explore Methods: The expression levels of miR-34b in bladder cancer BIU-87 cells, SV-HUC-1 cells and renal tubular epithelial HK-2 cells were determined by real-time fluorescent quantitative PCR. After transfection with miR-34b mimic, the proliferation rate and clone formation rate of BIU-87 cells were detected by MTT assay and clone formation assay, respectively. After co-transfection with miR-34b mimic and ubiquitin-specific peptidase 22 (USP22)-wide type (WT) or USP22-mutation type (Mut), the specific binding ability of miR-34b to 3'-untranslated region (3'-UTR) in USP22 gene was examined by luciferase reporter system. The expression levels of USP22 mRNA and protein in BIU-87 cells after transfection with miR-34b mimic were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. Results: The expression level of miR-34b in human bladder cancer BIU-87 cells was lower than those in SV-HUC-1 cells and renal tubular epithelial HK-2 cells (both P < 0.05). The proliferation rate and clone formation rate of BIU-87 cells in miR-34b mimic transfection group were lower than those of blank control cells (BIU-87 cells without any transfection) and negative control cells [BIU-87 cells transfected with miRNA mimic negative control (miRNA mimic NC)] (all P < 0.05). The result of luciferase reporter system indicated that miR-34b targeted 3'-UTR in USP22 gene. The luciferase activity of BIU-87 cells in co-transfection with miR-34b mimic and recombinant vector USP22-WT group was decreased (P < 0.05). The expression level of USP22 protein in BIU-87 cells in miR-34b mimic transfection group were lower than those in the blank control and the negative control groups (all P < 0.05). Conclusion: miR-34b can suppress the proliferation of bladder cancer BIU-87 cells. This effect may be related to the expression of USP22.