Liu Yue, Pei Shengfei, Wang Xue, Li Xueying, Long Yifei, Sun Shufeng, Meng Chunyan, Feng Fumin
School of Public Health, North China University of Science of Technology, Tangshan, 062310, Hebei, China.
College of Nursing and Rehabilitation, North China University of Science and Technology, Tangshan, Hebei Province, China.
Mol Biotechnol. 2024 Dec 23. doi: 10.1007/s12033-024-01355-8.
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.
乙型肝炎是由乙型肝炎病毒（HBV）引起的肝脏病毒感染。恩替卡韦（ETV）被认为是治疗HBV的主要治疗选择，主要通过抑制HBV复制发挥作用。泛素特异性肽酶7（USP7）是一种去泛素化酶，在调节DNA修复机制中起关键作用。本文旨在研究USP7在HBV复制中的作用及其增强ETV抗病毒疗效的潜力，同时探索其中涉及的潜在机制。HBV感染与肝癌的发生密切相关。在本研究中，我们选择了HepG2.2.15细胞系，它是稳定转染了两个完整HBV基因组的HepG2细胞。HepG2.2.15支持HBV的复制、组装和分泌。随后采用定量实时聚合酶链反应（qRT-PCR）和蛋白质免疫印迹（WB）分析来测量两种细胞系中USP7的mRNA和蛋白质水平。使用小干扰RNA（siRNA）使USP7基因沉默，使用Lipofectamine 6000™转染试剂将siRNA-USP7转染到细胞中，之后我们在HepG2.2.15细胞中培养24、48和72小时后评估HBV复制、HBsAg和HBeAg的水平。之后，将HepG2.2.15细胞分为几组：对照组、通过siRNA沉默USP7基因的组（siRNA-USP7）、USP7沉默阴性对照组（siRNA-NC）、ETV药物治疗组（ETV）、ETV药物治疗联合通过siRNA沉默USP7基因的组（ETV + siRNA-USP7）以及ETV治疗联合siRNA沉默阴性对照组（ETV + siRNA-NC）。培养24、48和72小时后评估细胞上清液中的HBV复制、HBsAg和HBeAg水平。此外，测量丙氨酸氨基转移酶（ALT）和天冬氨酸氨基转移酶（AST）水平以评估细胞损伤。此外，利用qRT-PCR和蛋白质免疫印迹技术分析p53的mRNA和蛋白质水平作为USP7潜在下游机制，同时评估p53信号通路中Bax和Bcl-2的mRNA和蛋白质水平。最后，我们通过免疫共沉淀研究USP7与p53蛋白之间的相互作用。在HepG2.2.15细胞系中USP7蛋白和mRNA水平上调，沉默USP7可抑制HBV复制。更重要的是，与其他组相比，ETV + siRNA-USP7组中的HBV复制、HBsAg和HBeAg水平显著降低（P < 0.05），表明沉默USP7可增强ETV的抗病毒效果。此外，ALT和AST水平显著降低（P < 0.05），表明细胞损伤减少。此外，观察到USP7与p53之间存在相互作用。ETV + siRNA-USP7组中p53及其下游因子Bax和Bcl-2的mRNA和蛋白质水平均显著下调（P < 0.05），这意味着USP7参与调节p53通路。在人肝癌模型中降低去泛素化肽酶7的表达可增强恩替卡韦的抗病毒效果并减少乙型肝炎病毒引起的细胞损伤。
