新型 HBx 突变 F30V 与体内肝细胞癌相关，降低乙型肝炎病毒复制效率，并增强 HBx N 端的抗凋亡活性。
The novel HBx mutation F30V correlates with hepatocellular carcinoma in vivo, reduces hepatitis B virus replicative efficiency and enhances anti-apoptotic activity of HBx N terminus in vitro.
机构信息
Department of Experimental Medicine and Surgery, University of Rome 'Tor Vergata' Rome, Italy.
Department of Experimental Medicine and Surgery, University of Rome 'Tor Vergata' Rome, Italy; Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain.
出版信息
Clin Microbiol Infect. 2019 Jul;25(7):906.e1-906.e7. doi: 10.1016/j.cmi.2018.11.017. Epub 2018 Nov 23.
OBJECTIVE
We aimed to investigate HBx genetic elements correlated with hepatitis B virus (HBV) -related hepatocellular carcinoma (HCC) and their impact on (a) HBV replicative efficiency, (b) HBx binding to circular covalently closed DNA (cccDNA), (c) apoptosis and cell-cycle progression, and (d) HBx structural stability.
METHODS
This study included 123 individuals chronically infected with HBV: 27 with HCC (77.9% (21/27) genotype D; 22.1% (6/27) genotype A) and 96 without HCC (75% (72/96) genotype D; 25.0% (24/96) genotype A). HepG2 cells were transfected by wild-type or mutated linear HBV genome to assess pre-genomic RNA (pgRNA) and core-associated HBV-DNA levels, HBx-binding onto cccDNA by chromatin immunoprecipitation-based quantitative assay, and rate of apoptosis and cell-cycle progression by cytofluorimetry.
RESULTS
F30V was the only HBx mutation correlated with HCC (18.5% (5/27) in HCC patients versus 1.0% (1/96) in non-HCC patients, p 0.002); a result confirmed by multivariate analysis. In vitro, F30V determined a 40% and 60% reduction in pgRNA and core-associated HBV-DNA compared with wild-type (p <0.05), in parallel with a significant decrease of HBx binding to cccDNA and decreased HBx stability. F30V also decreased the percentage of apoptotic cells compared with wild-type (14.8 ± 6.8% versus 19.1 ± 10.1%, p <0.01, without affecting cell-cycle progression) and increased the probability of HBx-Ser-31 being phosphorylated by PI3K-Akt kinase (known to promote anti-apoptotic activity).
CONCLUSIONS
F30V was closely correlated with HBV-induced HCC in vivo, reduced HBV replicative efficiency by affecting HBx-binding to cccDNA and increased anti-apoptotic HBx activity in vitro. This suggests that F30V (although hampering HBV's replicative capacity) may promote hepatocyte survival, so potentially allowing persistent production of viral progeny and initiating HBV-driven hepatocarcinogenesis. Investigation of viral genetic markers associated with HCC is crucial to identify those patients at higher risk of HCC, who hence deserve intensive liver monitoring and/or early anti-HBV therapy.
目的
我们旨在研究与乙型肝炎病毒（HBV）相关肝细胞癌（HCC）相关的 HBx 遗传元件及其对以下方面的影响：（a）HBV 复制效率；（b）HBx 与环状共价闭合 DNA（cccDNA）的结合；（c）细胞凋亡和细胞周期进程；以及（d）HBx 结构稳定性。
方法
本研究纳入了 123 名慢性 HBV 感染者：27 名 HCC 患者（77.9%（21/27）为基因型 D；22.1%（6/27）为基因型 A）和 96 名无 HCC 患者（75%（72/96）为基因型 D；25.0%（24/96）为基因型 A）。通过野生型或突变线性 HBV 基因组转染 HepG2 细胞，评估前基因组 RNA（pgRNA）和核心相关 HBV-DNA 水平，通过基于染色质免疫沉淀的定量测定法评估 HBx 与 cccDNA 的结合，以及通过细胞荧光计评估细胞凋亡和细胞周期进程。
结果
F30V 是唯一与 HCC 相关的 HBx 突变（HCC 患者中为 18.5%（5/27），而非 HCC 患者中为 1.0%（1/96），p 0.002）；这一结果通过多变量分析得到证实。在体外，与野生型相比，F30V 导致 pgRNA 和核心相关 HBV-DNA 减少 40%和 60%（p <0.05），同时 HBx 与 cccDNA 的结合显著减少，HBx 稳定性降低。与野生型相比，F30V 还降低了细胞凋亡的百分比（14.8 ± 6.8%比 19.1 ± 10.1%，p <0.01，而不影响细胞周期进程），并增加了 PI3K-Akt 激酶磷酸化 HBx-Ser-31 的可能性（已知可促进抗凋亡活性）。
结论
F30V 与体内 HBV 诱导的 HCC 密切相关，通过影响 HBx 与 cccDNA 的结合降低了 HBV 的复制效率，并增加了体外抗凋亡 HBx 活性。这表明 F30V（尽管阻碍了 HBV 的复制能力）可能促进肝细胞存活，从而可能允许持续产生病毒产物并启动 HBV 驱动的肝癌发生。研究与 HCC 相关的病毒遗传标志物对于确定那些具有更高 HCC 风险的患者至关重要，这些患者因此需要进行更密集的肝脏监测和/或早期抗 HBV 治疗。
Zotero 插件