Effect Of Up-regulation Of Fibroblast Growth Factor 15/19 Signaling On Hepatocellular Carcinoma In High Fat Environment And Its Mechanism

Background and objective:Fibroblast growth factor(FGF)15/19 is a hormone-like regulatory factor that regulates bile acids,participates in glycolipid metabolism,and plays an important role in the development and progression of tumors.The protein encoded by the human FGF19 gene is similar to the protein encoded by the mouse FGF15 gene.FGF15 can be regarded as a homologous gene of FGF19.FGF15/19 is abundantly expressed in the distal small intestine,and its specific receptor FGFR4 and co-receptor β-Klotho binding further up-regulates the expression of cyclin D1 by activating Wnt/β-catenin signaling,which ultimately leads to the proliferation and growth of tumor cells.Hepatocellular carcinoma(HCC)is one of the most common malignant tumors in the clinic,and it is the fastest growing cause of cancer-related death.The treatment effect is not good,and it is urgent to study its pathogenesis.At present,it is believed that tumor micro-environment and cancer stem cells play an important role in the occurrence and development of liver cancer.Liver cancer is a metabolic disease.The relationship between metabolic changes and tumorigenesis and development has been clarified.However,its mechanism of affecting tumor cell development and development is still unclear.There is a correlation between nonalcoholic fatty liver disease(NAFLD)or nonalcoholic steatohepatitis(NASH)and HCC.A more comprehensive understanding of this disease mechanism is critical for early diagnosis,treatment,and even prevention.We tried to investigate the effect and mechanism of FGF15/19 signal upregulation on liver cancer through the NASH-HCC model.Methods:The NASH-HCC model was established in male C57L/J mice treated with N-nitrosodiethylamine(DEN)and a high fat diet(HFD).UT+CD in the control group,UT+HFD in the high-fat diet group,DEN+CD in the low-fat diet group,DEN+HFD in the high-fat diet group,biochemical analysis,ELISA,Western blot,RT-PCR,immunohistochemistry Staining,HE staining,immunofluorescence staining,si RNA,flow cytometry and other methods to study FGF15-FGFR4 signal changes,inflammation,lipid metabolism,cellular activities,epithelial-mesenchymal transition,and Wnt/ in mice The mechanism of action is discussed in the β-catenin signaling pathway.A series of studies were performed using FFA to treat Hapal-6 cells(mouse liver cancer cell lines),H4 IIE cells(rat liver cancer cell lines)and Hep G2 cells(human HCC cell lines).Finally,the results of in vivo and in vitro experiments were also verified in NASH-HCC human HCC liver cancer specimens.Results:(1)Ultrasound was used to monitor the development of liver tumors in mice at different time points.The results showed that DEN+HFD group showed an invasive tumor growth pattern,and the gross anatomical results confirmed the results of the ultrasound examination.HE staining and oil red staining of liver tissue in mice showed the characteristics of steatohepatitis and liver cancer in DEN+HFD group,indicating the NASH-HCC model was build successfully.Body weight,liver weight,and triglyceride(TG)levels were significantly increased in DEN+HFD group,suggesting that lipid metabolism disorders may accelerate DEN-induced oncogenic transformation.Mice with high-fat diet had abnormal blood glucose metabolism and insulin resistance,especially in the high-fat group.It is suggested that metabolic disorders are associated with tumors.(2)After successful construction of the NASH-HCC mouse model,we examined the expression level of FGF15.The results showed that the expression of FGF15 in serum,ileum or liver tissue was significantly increased comparing with other groups.(3)The results showed that the expression of FGFR4,β-klotho and fatty acid synthase(FASN)was increased significantly measured in the experimental group(UT+HFD,DEN+CD and DEN+HFD)by IHC,Western blot.It suggests that liver cancer and high-fat environment can promote the expression of FGFR4 and β-klotho,and FGF15 signaling is involved in abnormal lipid synthesis during carcinogenesis of HCC.(4)HCC expresses stem cell markers through invasive features.Because epithelial cell adhesion molecule(EPPM)and CD133 are widely accepted surface markers in HCC,we further analyzed the expression of Ep CAM and CD133 to study their potential role in carcinogenic transformation process in NASH-HCC mice.The results showed that the number of Ep CAM-positive cells in the DEN+HFD group increased significantly.β-catenin is a key component of the Wnt pathway and is associated with Ep CAM.We further analyzed the protein expression levels of β-catenin.The results showed that β-catenin not only increased significantly in the tumor group,but also increased in the liver tissue of mice in the high-fat group.These results indicate that liver lipids Metabolism may play an important role in the pathogenesis of Ep CAM and Wnt/β-catenin signaling-mediated carcinogenesis.The above results were further verified in in vitro experiments,which support the development and progression of liver cancer by promoting FGF15 signaling by promoting EMT.(5)Blocking FGFR4 eliminates FFA-promoted migration and abnormal signaling.After treatment with FFA,the migration ability of Hepal-6 cells was significantly increased.After the inhibitor was used,the migration ability of Hepal-6 cells was blocked.Inhibition of FGFR4 significantly reduced FFA-induced Ep CAM,β-catenin and Cyclin D1 expression.To investigate whether silencing the key genes of the FFA signal would affect these downstream molecular events,Hapal-6 cells were treated with si PPAR-α and si CD36,and then the cells were treated with FFA.The results showed that the expression of Ep CAM,β-catenin and Cyclin D1 decreased after si RNA knockdown PPAR-α and CD36,and the interaction between FFA and FGF15/19-FGFR4 signaling promoted tumor initiation and liver cancer.(6)Abnormal signal transduction of FGF19 in human NASH-HCC.The results of IHC triple staining showed that FGF19/FGFR4/β-klotho were abundantly expressed anddistributed in liver cancer tissues at the same time.Significantly increased Ep CAM expression was also observed in liver cancer tissues compared to adjacent benign tissues.The expression of FGFR4,FASN,AFP and β-catenin was significantly up-regulated in tumors of patients with NASH-HCC compared with benign tissues.Conclusion:(1)The expression of FGF15/19 and FGFR4 and β-klotho is up-regulated in a high-fat environment,which together activates downstream signaling and plays a key role in the occurrence and development in HCC.(2)Liver lipid metabolism may play an important role in the pathogenesis of Ep CAM and Wnt/β-catenin signaling-mediated carcinogenesis.(3)Blocking FGFR4 can eliminate free fatty acid-promoted tumor migration ability and abnormal signaling,and is a potential therapeutic target.Further study of FGF15/19 signaling in a high-fat environment is important for understanding the pathogenesis of HCC.