Hepatitis B Virus X Protein Differentially Regulates Hepatocellular Carcinoma Metastasis In Various Nutrient Conditions Via SIRT3 Protein

Background and Aim: Hepatocellular Carcinoma(HCC),harboring distinctly high incidence rate and cancer death rate,is characterized by prevalently coupled infection with Hepatitis B virus(HBV)in china.The expression of Hepatitis B virus X protein(HBx),an HBV-encoded protein with multiple functions,is supposed to contribute to the development of HCC.However,paradoxical effects of HBx on HCC are frequently reported,which mechanism remains unknown.We have reported that varied nutrient conditions lead to different HBx roles in Hep G2 apoptosis,driving us to speculate that the diversity of nutrient conditions is one of the main causes of the polymorphism of HBx effects.In this study,we focus on the heterogeneous roles and underlying mechanisms of HBx in HCC metastasis under various nutrient conditions,then explore ways to reverse the effect of HBx on promoting HCC metastasis and therapeutic methods to inhibit HBVrelated HCC metastasis.Methods: 1.Firstly,we collected tissues of human HBV-related HCC and analyzed tumor nutrient microenvironments as well as their relationships with HCC metastasis through immunohistochemitry with serial sectioning.2.At cellular level,heterogenous roles of HBx on metastasis of hepatoma cells under adequate nutrition or in stavation were evaluated by western blot,invasion and migration experiments and the underlying mechanism was explored with SIRT3 protein by genetic suppression of SIRT3.Moreover,the regulation of SIRT3 protein expression by HBx was intensively studied by real-time PCR,Co-IP and autophagy inhibitors.Additionally,the roles of SIRT3 in the growth and metastasis of hepatocarcinoma were verified with cultured cells,subcutaneous tumor model and liver-in-situ implantation metastasis model in nude mice.3.Next,the function of a new anti-hepatocarcinoma drug,trichostatin A,in HBx-related HCC metastasis under nutrient deprivation was characterized by western blot and the role of SIRT3 in this process was determined by western blot and real-time PCR.4.Finally,we adjusted glucose concentrations in cell culture medium to drive HBx to inhibit HCC metastasis,verified by western blot,cell-based glucose uptake detection,ROS detection,invasion and migration test;and we further enhanced hepatic glucose uptake in orthotopic HCC mice model by insulin injection plus glucose intake to inhibit HBV-related HCC metastasis.Results: 1.Hunman HBV-related HCC tissues exhibits various nutrient microenvironments even in same section,and the starvation-evoked protein marker is related to tumor metastasis markers and SIRT3 expression.2.At cellular level,HBx differentially regulates the migration and invasion of hepatoma cells through diversely adjusting the expression of SIRT3.Simultaneously,we find that autophagy pathway contributes to the degradation of SIRT3 protein,and HBx plays a role in this degradation.Additionally,we has verified the suppressive effect of SIRT3 on HCC metastasis.3.Next,we show that trichostatin A upregulates the SIRT3 mRNA levels to inhibit HBxrelated HCC metastasis in nutrient-deprived condition.4.Finally,we address that extremely increasing glucose concentration drives HBx to inhibit HCC metastasis,and substantially enhancing the hepatic glucose uptake in mice efficiently reduces HBV-related HCC metastasis.Conclusions: 1.HBx differentially adjusts HCC invasion and metastasis under varied nutrient conditions through regulating autophagy-related SIRT3 protein degradation.2.And extremely increasing the glucose uptake of hepatoma cells efficiently drives HBx to play a suppressive role in HCC metastasis and inhibits the metastasis of HBV-related HCC.3.Additionally,Trichostatin A efficiently inhibits HBx-related HCC metastasis in starvation environment by upregulating SIRT3 transcripts.