Differ-omics Of The Major Isoforms Of Human Nrf1 From Nrf2 By Profiling Their Regulatory Spectrums And Their Significances Of Application In HCC

Nrf1 and Nrf2,two principal members of the CNC-b ZIP transcription factor family with highly conserved functional domains,are essential for maintaining normal development and steady-state balance of the organism.However,there exist many isoforms with different activities of Nrf1,and some regulatory functions of this factor overlap with or differ from those of Nrf2.This may make it difficult to distinguish their respective regulated gene sets,biological processes and corresponding function mechanisms.Therefore,it is of great scientific significance to establish effective research tools specific for distinct Nrf1 isoforms and Nrf2,and reveal those different regulatory profiles mediated by Nrf1 isoforms and Nrf2,along with mapping relationships between these two transcription factors and relevant biological phenomena,for exploring more clinical applicable potentials of Nrf1 and Nrf2.Hepatocellular carcinoma（HCC）,a primary liver cancer with high mortality in clinic,has suffered from a lack of effective treatment strategies for targeting its complex pathogenesis.Specific knockout of nrf1 in mouse liver spontaneously results in a phenotype of primary hepatoma.Knockout of Nrf1-derived long isoforms in the human hepatoma cells leads to aberrant hyper-activation of Nrf2 and promotes the malignant growth of subcutaneous tumor xenograft in nude mice,whilst knockout of Nrf2 inhibits the malignant growth of xenograft tumors.This implies that a crosstalk between Nrf1and Nrf2 exerts a significant role in progression of HCC.However,their inter-regulatory network and differential application in HCC remain to be identified.To address these scientific issues,a set of the controllable modes cell lines for stably expressing each of Nrf1 main isoforms or Nrf2 have been established herein.These cell lines were employed together with the other hepatoma cell lines with gene-editing of either Nrf1α/TCF11 or Nrf2,which had been previously constructed in our laboratory,as the research materials in this thesis.These research materials were subjected to multiple omics analysis and different levels of biological experiments,aiming to explore those putative similarities and differences among the regulatory profiles of Nrf1 main isoforms and Nrf2,their inter-regulatory network and differential potential applications in HCC.Thus this study will provide an important scientific basis and another reference for distinguishing Nrf1 isoform-specific functions,clarify the relevant and different emphases between the functions of Nrf1α/TCF11 and Nrf2,and develop a potential strategy for chemoprevention and treatment of HCC by intervening the certain interactive regulation system governed by Nrf1 and Nrf2.The relevant results of this study are as shown below:（1）The controllable model cell lines for stably expressing TCF11,TCF11^ΔN,Nrf1α,Nrf1β,Nrf1γor Nrf2,respectively,were established on the base of Flp-In^TMT-REx^TM-293 system herein.（2）The regulatory profiles of Nrf1 and Nrf2 with their targets were thoroughly analyzed and scrutinized.The regulatory range of each Nrf1 isoform is affected by its transactivation region and its NTD domain that negatively regulates its trans-activity.The similar but different regulatory modes of each Nrf1 isoform on target genes could enhance the plasticity of Nrf1 regulatory ability.In normal cells,Nrf1 prefers to regulate carbohydrate metabolism homeostasis,macromolecular transport and localization process,and the process of cardiovascular diseases more than Nrf2.Whilst Nrf2 prefers to regulate the organism development process,the progression of neurodegenerative diseases and immune diseases more than Nrf1.Notably,both of Nrf1 and Nrf2 can regulate gene transcription and expression,stress response in response to various stimulus,signal transduction process,macromolecular metabolism process,cell growth and death process,the progression of infectious diseases and cancer,but their regulation modes in the same biological process appear to be almost opposite.In addition,the regulatory function and regulatory mode of TCF11^ΔN,which was derived from the deletion of the NTD domain that anchors the endoplasmic reticulum and its adjacent juxtamembrane proteolytic degron from TCF11,seems to be rather similar to that of Nrf2.TCF11^ΔN could be regarded as a reference model isoform for Nrf1 to resuce Nrf2’s function.（3）The associated regulatory network regulated by Nrf1α/TCF11 and Nrf2 is mainly responsive to various exogenous or endogenous stimulus and activates PI3K-Akt,VEGF and other relevant signal transduction pathways by regulating signal molecules and their interactions,aiming to regulate the metabolism of carbohydrate,lipid and amino acids,cell proliferation,migration and survival of hepatoma cells,so as to control the progression of HCC.In hepatoma cells,Nrf1α/TCF11 prefer to regulate the Jak-STAT signaling pathway,inositol phosphate metabolism,betaine metabolism,cysteine and methionine metabolism more than Nrf2.Whilst Nrf2 prefers to regulate the Wnt signaling pathway,steroid biosynthesis,glutathione metabolism and purine metabolism more than Nrf1α/TCF11.Furtherly,Nrf1α/TCF11 could also govern Nrf2to regulate its downstream pathway in the pathological process of hepatoma.（4）TCF11 is more effective than Nrf1αin inhibiting the malignant proliferation and migration of hepatoma cells,and preventing the malignant growth of tumor xenografts transplanting hepatoma cells subcutaneously in nude mice.Both TCF11 and Nrf1αcan induce tumor coagulative necrosis and up-regulate the proteins which are beneficial to the prognostic survival of HCC,thus they are expected to be new strategic targets for prevention and treatment of HCC.By contrast,Nrf2 exerts dual positve and negative roles in the regulation of proteins associated with the prognostic survival of HCC,and such dual effects are also related to the malignancy of hepatoma cells.However,Nrf2 still has a tendency to form an interactive regulation system with Nrf1 to intervene the pathogenesis of HCC,thereby regarded as an assistant target to Nrf1α/TCF11 in the chemoprevention and treatment of HCC.Altogether,these inter-regulatory profiles of Nrf1 and Nrf2 enable for maintaining cell homeostasis and orderly operation of the organism to a greater extent.The multiple regulatory system composed of Nrf1α/TCF11 and Nrf2,along with functional genes involved in their joint regulatory network,play an important role in their defense against the malignant progression of HCC.