Hepatitis B is a viral infection of the liver caused by the hepatitis B virus (HBV). Entecavir (ETV) is considered the primary therapeutic option for HBV treatment, primarily functioning by inhibiting HBV replication. Ubiquitin-specific peptidase 7 (USP7), a deubiquitinating enzyme, plays a crucial role in regulating DNA repair mechanisms. This article aims to investigate the role of USP7 in HBV replication and its potential to enhance the antiviral efficacy of ETV, while exploring the underlying mechanisms involved. HBV infection is closely associated with the development of liver cancer. In this study, we selected the HepG2.2.15 cell line, which was stably HepG2 cell transfected with two complete HBV genomes. HepG2.2.15 supports HBV replication, assembly, and secretion. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) assays were subsequently employed to measure USP7 mRNA and protein levels in both cell lines. The USP7 gene was silenced using small interfering RNA (siRNA), cells were transfected with siRNA-USP7 using Lipo6000™ Transfection Reagent, after which we assessed HBV replication, the levels of HBsAg, and HBeAg following 24, 48, and 72 h of culture in HepG2.2.15 cells. Afterwards, HepG2.2.15 cells were divided into several groups: control, USP7 gene silencing by siRNA group (siRNA-USP7), USP7 silencing negative control group (siRNA-NC), ETV drug treatment (ETV), ETV drug treatment combined with USP7 gene silencing by siRNA group (ETV + siRNA-USP7), and ETV therapy alongside a negative control for siRNA silencing (ETV + siRNA-NC). HBV replication, the levels of HBsAg, and HBeAg in the cell supernatant were assessed after 24, 48, and 72 h of culture. Additionally, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured to evaluate cellular damage. Furthermore, qRT-PCR and Western blot techniques were utilized to analyze p53 mRNA and protein levels as potential downstream mechanisms of USP7, along with assessing Bax and Bcl-2 mRNA and protein levels within the p53 signaling pathway. Lastly, we investigated the interaction between USP7 and p53 proteins through co-immunoprecipitation. USP7 protein and mRNA levels were up-regulated in the HepG2.2.15 cell line, and silencing of USP7 inhibited HBV replication. More importantly, HBV replication, HBsAg, and HBeAg levels in the ETV + siRNA-USP7 group were significantly reduced compared to the other groups (P < 0.05), indicating that silencing USP7 enhances the antiviral effect of ETV. Additionally, ALT and AST levels were significantly decreased (P < 0.05), suggesting a reduction in cellular damage. Furthermore, an interaction between USP7 and p53 was observed. Both mRNA and protein levels of p53, as well as its downstream factors Bax and Bcl-2 in the ETV + siRNA-USP7 group, were significantly down-regulated (P < 0.05), implying that USP7 is involved in regulating the p53 pathway. Decreasing of deubiquitinating peptidase 7 expression in a human hepatoma model enhanced antiviral effect of entecavir and reduced cellular damage caused by the hepatitis B virus.
Keywords: Antiviral; Deubiquitinating peptidase 7 (USP7); Deubiquitination; Entecavir (ETV); Hepatitis B virus (HBV).
© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.