NF-κB 和 miR-138-5p 对 APOBEC3B 诱导和 cccDNA 衰变的控制

StephenW

NF-κB 和 miR-138-5p 对 APOBEC3B 诱导和 cccDNA 衰变的控制
Suzanne Faure-Dupuy 1 2 , Tobias Riedl 1 3 , Maude Rolland 4 , Zoheir Hizir 4 , Florian Reisinger 1 5 , Katharina Neuhaus 1 , Svenja Schuehle 1 3 , Caroline Remouchamps 4 , Scheler 1 7 Schuehler 1 7 尼古拉斯·吉勒·米拉西莱特3、Jochen Wettengel 5、Romain Barnault 8、Romain Parent 8、Linda Christina Schuster 9、Rayan Farhat 8、Sandra Prokosch 1、Corinna Leuchtenberger 1、Rupert Öllinger 10、Thomas Engleitner 10、Karsten 10、Karsten 10 ，Darjus Tscharahganeh 12，Daniel B Lipka 7 13，Ulrike Protzer 5，David Durantel 8，Julie Lucifora 8，Emmanuel Dejardin 4，Mathias Heikenwälder 1 2
隶属关系
隶属关系

    1
    德国海德堡德国癌症研究中心 (DKFZ) 慢性炎症和癌症科。
    2
    德国海德堡海德堡大学传染病分子病毒学系。
    3
    德国海德堡海德堡大学生物科学学院。
    4
    比利时列日大学 GIGA 研究所分子免疫学和信号转导实验室。
    5
    德国慕尼黑 Helmholtz Zentrum München 病毒学研究所。
    6
    比利时那慕尔那慕尔生命科学研究所综合兽医研究组。
    7
    部分转化癌症表观基因组学，转化医学肿瘤学部，德国癌症研究中心 (DKFZ) 和国家肿瘤疾病中心 (NCT)，德国海德堡。
    8
    INSERM，U1052，里昂癌症研究中心 (CRCL)，里昂大学 (UCBL1)，CNRS UMR_5286，法国里昂莱昂贝拉尔中心 (CLB)。
    9
    德国癌症研究中心 (DKFZ) 和德国海德堡 Bioquant 染色质网络部。
    10
    德国慕尼黑 Rechts der Isar 大学医院分子肿瘤学和功能基因组学研究所。
    11
    德国 Neuherberg Helmholtz Zentrum München 辐射细胞遗传学研究室。
    12
    亥姆霍兹大学小组“细胞可塑性和表观遗传重塑”，德国癌症研究中心 (DKFZ) 和德国海德堡大学医院病理学研究所。
    13
    德国马格德堡 Otto-von-Guericke 大学医学院。

    PMID：34704004 PMCID：PMC8523871 DOI：10.1016/j.jhepr.2021.100354

抽象的

背景和目的：免疫介导的胞苷脱氨酶 APOBEC3B (A3B) 表达诱导导致 HBV 共价闭合环状 DNA (cccDNA) 衰变。在这里，我们旨在破译 A3B 诱导和相关 HBV 控制中涉及的信号通路和调节机制。

方法：分化的 HepaRG 细胞（dHepaRG）敲低了 NF-κB 信号成分，用 siRNA 或微 RNA（miRNA）转染，原代人肝细胞 ± HBV 或 HBVΔX 或 HBV-RFP，用淋巴毒素β受体（LTβR）处理-激动剂 (BS1)。通过逆转录酶-qPCR、免疫印迹、荧光素酶活性、染色质免疫沉淀、电泳迁移率变化测定、靶向亚硫酸氢盐-、miRNA-、RNA-、基因组测序和质谱法分析生物学结果。

结果：我们发现经典和非经典 NF-κB 信号通路对于 A3B 诱导和抗 HBV 作用是必需的。免疫介导的 A3B 产生的程度与 A3B 启动子去甲基化无关，但受 miRNA 138-5p 表达 (hsa-miR-138-5p) 的转录后控制，促进 A3B mRNA 衰减。 Hsa-miR-138-5p 过表达降低了 A3B 水平及其抗病毒作用。值得注意的是，已建立的感染通过 A3B 启动子的表观遗传调节抑制了 BS1 诱导的 A3B 表达。用 LTβR 特异性激动剂 BS1 治疗 12 天足以将 cccDNA 库减少 80%，而不会对癌症相关宿主基因的子集造成显着损害。有趣的是，A3B 介导的对 HBV 的影响与 cccDNA 的转录活性以及 rcDNA 合成无关。

结论：总而言之，A3B 代表了唯一描述的针对转录活性和非活性 cccDNA 的酶。因此，在针对 HBV 的新疗法的组合设计中应考虑抑制 hsa-miR-138-5p 表达，尤其是在免疫介导的 A3B 诱导的背景下。

小结：免疫介导的胞苷脱氨酶 APOBEC3B 诱导受 NF-κB 信号的转录调控，并受 hsa-miR-138-5p 表达的转录后下调，导致 cccDNA 衰减。及时控制的 APOBEC3B 介导的 cccDNA 衰变与 cccDNA 转录活性无关，并且不会对癌症相关基因的子集造成损害。因此，APOBEC3B 介导的 cccDNA 衰减可以为靶向乙型肝炎病毒慢性感染提供有效的治疗替代方案。

关键词：A20，肿瘤坏死因子α诱导蛋白3； APOBEC3A/A3A，载脂蛋白B mRNA

StephenW

  Control of APOBEC3B induction and cccDNA decay by NF-κB and miR-138-5p
Suzanne Faure-Dupuy  1   2 , Tobias Riedl  1   3 , Maude Rolland  4 , Zoheir Hizir  4 , Florian Reisinger  1   5 , Katharina Neuhaus  1 , Svenja Schuehle  1   3 , Caroline Remouchamps  4 , Nicolas Gillet  6 , Maximilian Schönung  3   7 , Mira Stadler  1   3 , Jochen Wettengel  5 , Romain Barnault  8 , Romain Parent  8 , Linda Christina Schuster  9 , Rayan Farhat  8 , Sandra Prokosch  1 , Corinna Leuchtenberger  1 , Rupert Öllinger  10 , Thomas Engleitner  10 , Karsten Rippe  9 , Roland Rad  10 , Kristian Unger  11 , Darjus Tscharahganeh  12 , Daniel B Lipka  7   13 , Ulrike Protzer  5 , David Durantel  8 , Julie Lucifora  8 , Emmanuel Dejardin  4 , Mathias Heikenwälder  1   2
Affiliations
Affiliations

    1
    Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany.
    2
    Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
    3
    Faculty of Biosciences, Heidelberg University, Heidelberg, Germany.
    4
    Laboratory of Molecular Immunology and Signal Transduction, GIGA-Institute, University of Liège, Liège, Belgium.
    5
    Institute of Virology, Helmholtz Zentrum München, Munich, Germany.
    6
    Integrated Veterinary Research Unit, Namur Research Institute for Life Sciences, Namur, Belgium.
    7
    Section Translational Cancer Epigenomics, Division of Translational Medical Oncology, German Cancer Research Center (DKFZ) and National Center for Tumor Diseases (NCT), Heidelberg, Germany.
    8
    INSERM, U1052, Cancer Research Center of Lyon (CRCL), University of Lyon (UCBL1), CNRS UMR_5286, Centre Léon Bérard (CLB), Lyon, France.
    9
    Division of Chromatin Networks, German Cancer Research Center (DKFZ) and Bioquant, Heidelberg, Germany.
    10
    Institute of Molecular Oncology and Functional Genomics, Rechts der Isar University Hospital, Munich, Germany.
    11
    Research Unit of Radiation Cytogenetics, Helmholtz Zentrum München, Neuherberg, Germany.
    12
    Helmholtz-University Group 'Cell Plasticity and Epigenetic Remodeling', German Cancer Research Center (DKFZ) and Institute of Pathology University Hospital, Heidelberg, Germany.
    13
    Faculty of Medicine, Otto-von-Guericke-University, Magdeburg, Germany.

    PMID: 34704004 PMCID: PMC8523871 DOI: 10.1016/j.jhepr.2021.100354

Abstract

Background & aims: Immune-mediated induction of cytidine deaminase APOBEC3B (A3B) expression leads to HBV covalently closed circular DNA (cccDNA) decay. Here, we aimed to decipher the signalling pathway(s) and regulatory mechanism(s) involved in A3B induction and related HBV control.

Methods: Differentiated HepaRG cells (dHepaRG) knocked-down for NF-κB signalling components, transfected with siRNA or micro RNAs (miRNA), and primary human hepatocytes ± HBV or HBVΔX or HBV-RFP, were treated with lymphotoxin beta receptor (LTβR)-agonist (BS1). The biological outcomes were analysed by reverse transcriptase-qPCR, immunoblotting, luciferase activity, chromatin immune precipitation, electrophoretic mobility-shift assay, targeted-bisulfite-, miRNA-, RNA-, genome-sequencing, and mass-spectrometry.

Results: We found that canonical and non-canonical NF-κB signalling pathways are mandatory for A3B induction and anti-HBV effects. The degree of immune-mediated A3B production is independent of A3B promoter demethylation but is controlled post-transcriptionally by the miRNA 138-5p expression (hsa-miR-138-5p), promoting A3B mRNA decay. Hsa-miR-138-5p over-expression reduced A3B levels and its antiviral effects. Of note, established infection inhibited BS1-induced A3B expression through epigenetic modulation of A3B promoter. Twelve days of treatment with a LTβR-specific agonist BS1 is sufficient to reduce the cccDNA pool by 80% without inducing significant damages to a subset of cancer-related host genes. Interestingly, the A3B-mediated effect on HBV is independent of the transcriptional activity of cccDNA as well as on rcDNA synthesis.

Conclusions: Altogether, A3B represents the only described enzyme to target both transcriptionally active and inactive cccDNA. Thus, inhibiting hsa-miR-138-5p expression should be considered in the combinatorial design of new therapies against HBV, especially in the context of immune-mediated A3B induction.

Lay summary: Immune-mediated induction of cytidine deaminase APOBEC3B is transcriptionally regulated by NF-κB signalling and post-transcriptionally downregulated by hsa-miR-138-5p expression, leading to cccDNA decay. Timely controlled APOBEC3B-mediated cccDNA decay occurs independently of cccDNA transcriptional activity and without damage to a subset of cancer-related genes. Thus, APOBEC3B-mediated cccDNA decay could offer an efficient therapeutic alternative to target hepatitis B virus chronic infection.

Keywords: A20, tumour necrosis factor alpha-induced protein 3; APOBEC3A/A3A, apolipoprotein B mRNA

StephenW

https://www.jhep-reports.eu/article/S2589-5559(21)00130-0/pdf