Hepatitis B Virus X Protein Increases 8-Oxo-7,8-Dihydro-2?-Deoxyguanosine (8-Oxodg) Level via Repressing MTH1/MTH2 Expression in Hepatocytes
Hepatitis B Virus X Protein Increases 8-Oxo-7,8-Dihydro-2?-Deoxyguanosine (8-Oxodg) Level via Repressing MTH1/MTH2 Expression in Hepatocytes
复制标题
乙型肝炎病毒 X 蛋白通过抑制肝细胞中 MTH1/MTH2 表达来增加 8-Oxo-7,8-二氢-2?-脱氧鸟苷 (8-Oxodg) 水平
DOI：
10.1159/000495166
复制
发表时间：
2018-01-01
影响因子：
--
通讯作者：
Lin, Xu
中科院分区：
文献类型：
--
作者：
Lin, Yan-Ting;Liu, Wei;Lin, Xu
背景/目的：慢性B型肝炎病毒（HBV）感染显著增加了肝细胞癌（HCC）的发生风险。在HBV编码的七种病毒蛋白中，HBVX蛋白（HBx）似乎具有最大的致癌潜力。肝细胞内的HBx可诱导氧化应激，导致大量活性氧（ROS）的产生。高水平的ROS通常诱导氧化性DNA损伤和8-羟基-2-脱氧鸟苷（8-OHdG），也称为8-氧代-7，8-二氢-2-脱氧鸟苷（8-oxodG），其是DNA氧化的主要产物之一，也是氧化应激和致癌的重要生物标志物。细胞已经进化出一种机制，通过MTH 1（NUDT 1）、MTH 2（NUDT 15）、MTH 3（NUDT 18）和NUDT 5的核苷酸库消毒酶来防止氧化核苷酸掺入DNA。然而，很少有人知道HBx是否可以调节这些酶的表达和调节肝细胞中8-oxodG的形成和积累。研究方法：通过ELISA在稳定的HBV产生的肝癌细胞系、HBV感染小鼠模型、HBV和HBx转基因小鼠和HBV感染患者中相对于其各自的对照评估8-oxodG的水平。通过实时定量PCR和蛋白质印迹分析确定MTH 1、MTH 2、MTH 3和NUDT 5的表达。MTH 1和MTH 2表达的转录调控HBx和MTH 1和MTH 2启动子超甲基化的HBx的影响进行了研究，使用荧光素酶报告基因分析和亚硫酸氢盐测序分析。结果如下：与对照组相比，在稳定的HBV产生的HepG2.2.15细胞的基因组和培养上清液中，在HBV感染小鼠和HBV或HBx转基因小鼠的血清和肝组织中，以及在HBV感染患者的血清中，检测到显著更高水平的8-oxodG。肝细胞中HBx的表达显著增加8-oxodG水平，并在mRNA和蛋白水平上降低MTH 1和MTH 2的表达。研究还表明，HBx通过高甲基化显著减弱MTH 1或MTH 2启动子活性。此外，通过MTH 1和MTH 2的过表达，HBx对8-oxodG产生的增强是可逆的。结论：我们的数据显示，HBx表达通过抑制MTH 1和MTH 2的表达导致8-oxodG在肝细胞中的积累。这可能暗示HBx可能通过促进8-oxodG的突变潜力而作为肿瘤促进剂，从而将HBV感染和肝癌发生之间的可能联系联系起来。
Background/Aims: Chronic hepatitis B virus (HBV) infection markedly increases the risk of development of hepatocellular carcinoma (HCC). Among the seven viral proteins that HBV encodes, HBV X protein (HBx) appears to have the most oncogenic potential. The mitochondria-associated HBx can induce oxidative stress in hepatocytes, leading to the production of abundant reactive oxygen species (ROS). High levels of ROS usually induce oxidative DNA damage and 8-hydroxy-2-deoxyguanosine (8-OHdG), also known as 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG), which is one of the major products of DNA oxidation and an important biomarker for oxidative stress and carcinogenesis. Cells have evolved a mechanism to prevent oxidized nucleotides from their incorporation into DNA through nucleotide pool sanitization enzymes of MTH1 (NUDT1), MTH2 (NUDT15), MTH3 (NUDT18) and NUDT5. However, little is known as to whether HBx can regulate the expression of those enzymes and modulate the formation and accumulation of 8-oxodG in hepatocytes. Methods: The level of 8-oxodG was assessed by ELISA in stable HBV-producing hepatoma cell lines, an HBV infectious mouse model, HBV and HBx transgenic mice and HBV-infected patients versus their respective controls. Expression of MTH1, MTH2, MTH3 and NUDT5 was determined by a real-time quantitative PCR and western blot analysis. Transcriptional regulation of MTH1 and MTH2 expression by HBx and the effect of HBx on MTH1 and MTH2 promoter hypermethylation were examined using a luciferase reporter assay and bisulfite sequencing analysis. Results: In comparison with controls, significantly higher levels of 8-oxodG were detected in the genome and culture supernatant of stable HBV-producing HepG2.2.15 cells, in the sera and liver tissues of HBV infectious mice and HBV or HBx transgenic mice, and in the sera of HBV-infected patients. Expression of HBx in hepatocytes significantly increased 8-oxodG level and reduced the expression of MTH1 and MTH2 at both mRNA and protein levels. It was also demonstrated that HBx markedly attenuated the MTH1 or MTH2 promoter activities through hypermethylation. Furthermore, enhancement of 8-oxodG production by HBx was reversible by overexpression of MTH1 and MTH2. Conclusion: Our data show that HBx expression results in the accumulation of 8-oxodG in hepatocytes through inhibiting the expression of MTH1 and MTH2. This may implicate that HBx may act as a tumor promoter through facilitating the mutational potential of 8-oxodG thus connecting a possible link between HBV infection and liver carcinogenesis.