Nrf1/2管控脂肪肝炎转化肝癌进程中代谢信号-基因调控网络重编程的机制研究

The proposed project is integrated on the basis of the previously accumulated achievements from different groups and their preliminary work for this study. This proposal is orientated from basic research to clinical translation, with various strengths of interdisciplinary sciences being integrated. The objective is to create inducible knockout of Nrf1-/-:Nrf2-/- in the liver of mouse models that will develop with the phenotype of non-alcoholic steatohepatitis (NASH) and deterioratively transformed to hepatoma. The research platform is supplemented with relevant clinical specimens and the human cell lines that will be established by CRISPR/Cas9- and Talens--based gene editing to knockout Nrf1, Nrf2 and/or their subunits. These manufactures are combined with modern omics, RNA sequencing and systems biology approaches to analyze high-throughput data. The aims of this study will be focused to : i) identify which the metabolic signal transduction is directed by Nrf1 and/or Nrf2, that dictate target gene regulatory networks, and determine the mechanism by which the metabolic signal networks are reprogrammed in the course of NASH deterioration into liver cancer; ii) construct the "miRNA/lncRNA-transcription factors Nrf1/2- target gene expression networks " , and identify the key knots of the networks that enable to play important roles in the reprogramming of metabolic signals and their functionality in the mouse upon loss of Nrf1 and/or Nrf2 leading to steatohepatitis and its carcinogenesis ; iii) determine a crosstalk between key signaling pathways and lipid metabolism-regulatory networks in the mouse developing form the inflammation into carcinogenesis, and identify the mechanism underlying the action of crosstalk occurs in liver carcinogenesis Thereby, the project belongs to the forefront of the field to explore mechanisms by which transformation steatohepatitis into cancer is controlled by key molecular nodes within lipid metabolic signaling networks, aiming to establish a platform to screen novel drugs targeting for the prevention and treatment of inflammation-related cancer, in order to provide a novel strategy to develop a new technology targeting to cancer metabolism and relevant signaling from inflammation towards carcinogenesis.

本项目立足于前期工作，集成交叉科学优势，创建Nrf1:Nrf2双基因诱导敲除所致非酒精性脂肪肝炎癌变小鼠模型，辅以Talens或CRISPR/Cas9技术定点敲除人Nrf1/2亚型的细胞株和临床标本，结合现代组学测序及系统生物学分析，重点研究内容：i) 鉴定以Nrf1/2为轴心的代谢信号传导及其靶基因调控网络，并探讨该网络在脂肪肝炎恶化为肝癌病程中重编程的机制; ii) 构建miRNA/lncRNA-转录因子-基因表达的共调控网络，鉴定其关键分子对Nrf1/2敲除所致脂肪肝炎癌变进程中代谢信号重编程的作用机制; iii)阐明炎-癌链进程中关键信号通路与脂质代谢调控网络之间交叉对话及其在癌发生发展中的独特作用机制。该前沿机制研究的目标，旨在发掘脂肪肝炎-癌转化进程中代谢信号网络的关键节点，建立其靶向药物筛选平台，为炎癌转化防治提供新的策略思路，寻求靶向代谢攻击炎-癌转化细胞的新理论技术突破。

本项目立足于前期工作，集成交叉科学优势，成功构建了Nrf1Flox/flox: Cyp1A1Cre+/-、Nrf2-/-:Nrf1flox/flox:1A1-Cre小鼠疾病模型、以及Nrf1/Nrf2基因单敲除或双敲除细胞系。在此的基础上，结合临床相关疾病标本, 运用转录组学测序、代谢组及系统生物学分析, 深入研究Nrf1/Nrf2参与NASH恶性转化肝癌机制，而且取得了突破性新发现。其亮点成果: (1)模型动物NASH典型症状、过程可控又可逆，视为癌前病变恶化为肝癌的发生发展机制、以及抗癌前NASH药物研究的良好动物模型。(2)Nrf1敲除导致ER应激和SHH信号被激活，细胞自噬被抑制，对能量代谢相关信号网络也有复杂显著的影响。(3)除了hedgehog信号异常外，另一个监管途径Wnt/β-Catenin信号调控基因网络在其癌症恶性转化过程起重要作用。(4) Nrf1肝脏诱导敲除对Nrf2/ARE信号、以及ER应激、SHH、PI3K/Akt/mTOR和细胞自噬等信号网络的影响，确定了SHH信号在NASH和相关肿瘤细胞肝转移中的核心作用。(5)肝细胞中敲除Nrf1后则会引起脂质累积、炎症相关因子表达上调、肿瘤恶化。并在细胞水平上也成功建立NASH模型。（6）确定了Nrf1敲出后的相关表型主要通过Nrf2途径产生，而Nrf2敲除后肿瘤消失，且发现Nrf1与Nrf2之间存在着相互调控的关系。(7)另有组学数据分析，以阐明炎-癌链进程中关键信号通路与脂质代谢调控网络之间交叉对话及其在癌发生发展中的独特作用机制。(8）在该项目资助下，还发现了Nrf1表达的表观遗传学调控机制、Nrf2抑制因子Keap1的新型剪接变体、确定了Nrf1/2-mir22调控通路并构建了转录因子Nrf1和Nrf2共同下游和差异下游基因表达网络。总体来说，该项目的主要研究计划基本完成，并且建立了靶向药物筛选平台，为炎症癌症转化防治提供了新的研究策略和思路。