Long-term Hepatic MTORC1 Activation Causes Spontaneous Noninflammatory Steatotic Hepatocellular Carcinoma

BackgroundThe mammalian target of rapamycin(mTOR)pathway is aberrantly up-regulated in up to 50%of HCCs.There is an increasing number of HCC cases without inflammation and cirrhosis,especially in patients with nonalcoholic fatty liver disease,characterized only by hepatic steatosis.However,the molecular basis for the development of non-inflammatory HCC remains unclear..Previous study found that TSC1 knockout(LTsc1KO)mouse model spontaneously non-inflammatory,non-fibrosis HCC.Therefore,we want to use animal models to verify the possible mechanism of non-inflammatory HCC in vivo and its relationship with mTOR.Methods1.Use the Cre-loxp system to create hepatocyte specific TSC1 knockout mice.2.Verify knock out effect of the hepatocyte specific TSC1 knockout mice by PCR and western blot.3.Verify the hepatic-specific phenotypes demonstrated by LTsc1KO mice relative to control mice by HE staining and western blot.4.Analyze the liver inflammation in LTsc1KO mice by HE staining,immunohistochemistry,western blot and quantitative cytokine assays.5.Analyze the degree of hepatic fibrosis in LTsc1KO mice by sirius red staining,and the damage of hepatocytes by serum ALT and AST,in order to analyze the long-term chronic injury of the liver.6.Analyze the metabolic characteristics of LTsc1KO mice by metabolomics,oil red O staining and western blot.7.Detect the differential genes in LTsc1KO mice by RNA-seq and bioinformatic analysis,and validate by Q-PCR and western blot,in order to explore the molecular mechanism of HCC.8.Analyze the change of relevant factors and proteins after rapamycin gavage treatment.Statistical analysisContinuous variables were compared using independent sample t tests or one-way analysis of variance,with the data expressed as mean±SEM.Significant differences are indicated in the figure legends,unless otherwise indicated.Result1.Successfully created hepatocyte specific TSC1 knockout mice,the knockout effect was exactly,The phosphorylation of S6 was elevated in LTsc1KO mice tissues compared with control littermates.Moreover,the LTsc1KO mice developed spontaneous HCC2.Necrotic inflammation is not a necessary condition for spontaneous HCC in LTsc1KO mice,while characterized by significant lipid accumulation also displayed noncirrhotic and chemical carcinogenic characteristics.3.Hepatocarcinogenesis was subsequent to defects upstream and downstream of mTORC1 after TSC1 knock-down,reversed after rapamycin gavage,confirmed by western blot results.4.The expression of FGF21 wass getting lower and lower with the increasing of age,and it has been verified both at RNA level and protein level,indicated that the dysregulation of FGF21 was a later event for liver carcinogenesis in LTsc1KO mice.5.It was indicated that the crosstalk between mTORCl and the STAT pathway was linked to noninflammatory HCC in LTsc1KO mice,for the upregulation of anti-inflammatory cytokine STAT3 and downregulation of pro-inflammatory cytokine STAT5.6.The hepatic steatosis in LTsc1KO mice was mainly due to the low expression of protein regulating lipolysis and high expression of protein regulating lipid synthesis.Persistence of increased expression of SERBP1 was linked to lipid accumulation of HCC in LTsc1KO mice.Conclusion1.Inflammation and fibrosis are not prerequisites in HCC development triggered by LTsc1KO,while hepatic steatosis were manifested in HCC development.2.Complicated oncogenic network(Ddit4,Nuprl and FGF21)and mTORCl-STAT pathway crosstalk contributed to noninflammatory HCC.3.Up-regulated SERBP1 was linked to de novo lipid synthesis of steatotic HCC.