Study On The Role And Molecular Mechanism Of The Metabolic Nuclear Receptor FXR In Regulating Sox9~+ Cells Through Notch1

Hepatocellular carcinoma（HCC）is one of the most aggressive cancers with an increasing incidence.The aggressiveness of HCC is partially due to the presence of liver cancer stem cells（CSCs）,also known as tumor-initiating cells.Liver CSCs are highly tumorigenic and resistant to chemotherapy and radiation therapy,their presence is a major reason why HCC is difficult to treat.Farnesoid X receptor（FXR）has been shown to target CSCs and anti-tumor effects.In addition,FXR-knockout（FXR-KO）mice developed spontaneous HCC at the age of 12 months,which is consistent with the time to develop HCC in mice with constitutive activation of Notch1 reported in another previous study.Reports have suggested that Notch1 serves as the marker for oncogene and symmetric division.Like normal stem cells,CSCs also have the ability to divide symmetrically and asymmetrically.Unlike normal stem cells,CSCs have lost the ability to control their mode of cell division,resulting in continual excessive symmetric cell divisions and consequential uncontrolled tumor growth.However,the signaling pathway network involved in CSCs division remains elusive.Here,we aimed at exploring the role of FXR in regulation of the cell division of liver CSCs,and to provide the theoretical basis for targeted therapy of liver CSCs.The main results are as follows:1.Notch1 is involved in spontaneously developing liver tumors in FXR-KO mice.The expression of Notch1 and NICD1 nuclear translocation were detected by immunofluorescence assay or western bolt assay in the spontaneous liver tumor formation in FXR-KO mice.The results showed that the expression of Notch1 and nuclear translocation of NICD1 were increased in FXR-KO mice.2.The expression of FXR and Notch1 is inversely correlated in chronic liver injury.To investigate the relationship between FXR and Notch1,we constructed the DDC-induced or CCl₄-induced liver injury model in WT mice.The results showed that the expression of FXR was significantly decreased in the liver of DDC-fed mice,while the expression of Notch1 was significantly increased.Similar results were obtained in mice treated with CCl_4.3.FXR regulates asymmetric division of liver CSCs through Notch1.To investigate whether FXR regulates the expression of Notch1,Huh7 cells were treated with FXR ligand,followed by Quantitative real-time PCR（q RT-PCR）and WB to detect Notch1 expression.These results indicated that FXR activation repressed Notch1 expression.Subsequently,we examined whether FXR regulates the cell division of liver CSCs by pulse-chase Brd U labeling and paired-cell experiments,and found that FXR promoted asymmetric division of liver CSCs.Finally,we performed Notch1 overexpression or knocked-down the FXR experiment to explore whether asymmetric division of liver CSCs regulation by FXR was mediated through Notch1or not.The results showed that FXR induced asymmetric division of liver CSCs by inhibiting Notch1.4.Notch1 is a target gene of FXR.To determine whether Notch1 is a target gene of FXR,We explored whether Notch1 is a target gene of FXR by luciferase,EMSA and Ch IP assay.These results suggested that Notch1 is a target gene of FXR,and FXR inhibits Notch1 transcriptional activity by binding to its promoter.5.FXR directs asymmetric division of Sox9+cells and alleviates liver injury.In order to investigate whether FXR activation could regulate asymmetric division of Sox9⁺cells and expression of Notch1 in vivo.We constructed DDC-or CCl₄-induced chronic liver injury models in WT and FXR-KO mice,and treated them with GW4064.The results revealed that GW4064 could relieve DDC-or CCl₄-induced liver injury in WT mice,but not in FXR-KO mice.Meanwhile,the results also indicated that activation of FXR could increase the frequency of Sox9⁺cell asymmetric division and reduced Notch1 expression in WT mice,but not in FXR-KO mice.Conclusion:Our study reveals a critical role of FXR-Notch1 pathway in guiding the asymmetric division of liver CSCs,suggesting that this pathway may be exploited for the target therapy of liver CSCs.