Mechanisms By Which Transcription Factors Nrf1 And Nrf2 Differentially Regulate The Cellular Response To Er Stress And Lipid Metabolism In HCCs

In the long evolutionary process,eukaryotes have evolved a set of cytoprotection mechanisms to cope with the imbalance of cell homeostasis caused by external stimuli and/or internal abnormalities.Nrf1 and Nrf2,as two important members of the CNC-b ZIP protein family,have a highly conserved b ZIP domain,which can regulate the transcriptional activation of protective genes,such as those involved in antioxidant and detoxification,through binding of ARE or Ep RE,which is a key factor for maintaining homeostasis.Within endogenous regulation networks,Nrf1/Nrf2 can participate in the regulation of a variety of stress responses and metabolic processes,e.g.,endoplasmic reticulum(ER)stress and lipid metabolism.Nrf1 directly regulates transcriptional activation of the proteasomes and thus involved in UPR-related ERAD process.Lack of Nrf1 specifically in the murine livers leads to spontaneous development of NASH,and eventual deterioration into liver cancer.This proves that Nrf1 plays an important role in maintaining the ER steady-state and regulation of liver lipid metabolism.But,Nrf2 has been subjected to a even opposite way of regulation,in which PERK directly triggers the transcriptional activation of Nrf2.However,Nrf2-deficient cells exhibit a weakened ability to form tumor in nude mice.It is thus proved that Nrf1 and Nrf2 were not simply synergistic in the ER stress response,but also had certain differences in the lipid metabolism process.In this thesis,Nrf1/Nrf2 gene-editing cell lines were built on the base of the human liver cancer cell line Hep G2 and differently stimulated by TU,to analyze Nrf1/Nrf2 in regulation of ER stress mechanism for the unity of opposites.A variety of inhibitory or interfereing means were used to reduce or access the gene expression activity,in order to explore differentiated regulation of Nrf1/Nrf2 in molecular mechanisms of lipid metabolism.These main results were shown below:(1)The deletion of Nrf1 and Nrf2 leads to a decreased activity of ER stress-related genes and the poor responsiveness to higher concentrations of TU stimuled,which also shows a weakened responsive trend over a long period of time.This implies that Nrf1 and Nrf2 are essential for maintaining ER homeostasis.(2)Activation of Nrf1 can enhance the antagonistic ability of cells to respond to stimulation by TU in the presence or absence of Nrf2,but reduce activation of CHOP signalling and UPR-related apoptosis.This indicates that Nrf1 has a greater potential than Nrf2 at regulating ER stress responses.(3)Loss of Nrf1 leads to the disordered lipid metabolism in HCC cells.The lipid synthesis pathway was upregulated,but its decomposition pathway was partially down-regulated,while lipid droplets increased.By contrast,Nrf2 knockout cells give rise to decreased lipid synthesis and uptake capacity.These demonstrate that Nrf1 and Nrf2 contribute to significantly different regulation profiles of lipid metabolism.(4)Lipid deposition caused by knockout of Nrf1 can up-regulate the expression of CD36 by activating the PI3K-AKT-m TOR pathway and induce inflammatory response.Conversely,2BP can reduce lipid synthesis and lipid droplet production,and also lower the expression levels of CD36 and inflammatory signals.In conclusion from this thesis,I have explored the molecular mechanism by which Nrf1 and Nrf2 are unified in the regulation of ER stress response,along with differential regulation of lipid metabolism.This provides a theoretical approach to supporting for the prevention and treatment of related diseases by potentially targeting Nrf1/Nrf2.