HBx Mediated Increase Of SIRT1 Contributes To HBV-related Hepatocellular Carcinoma Tumorigenesis

Background:Hepatocellular carcinoma（HCC）is one of the main causes of cancer-related deaths worldwide,and chronic hepatitis B virus（HBV）infection is strongly associated with HCC development.The pathogenesis of hepatitis B related liver cancer（HBV-related HCC）remains obscure,but there is evidence that the viral protein HBx can promote the occurrence and development of HBV-related HCC by participating in host gene transcription,cell signal transduction,cell cycle and apoptosis.However,the molecular mechanism of HBx promoting HBV-related HCC has not been fully elucidated.Therefore,it is necessary to explore the mechanism of HBx in HBV-related HCC.Sirtuin 1（SIRT1）,as a member of sirtuins family,has been proved to enhance the replication of HBV;at the same time,many studies have found that SIRT1 can also promote the proliferation and metastasis of HCC.However,it is not clear whether SIRT1 can promote HCC by regulating HBx.To further explore the role of SIRT1 in HBV-related HCC and the potential relationship between SIRT1 and HBx will be helpful to analyze the mechanism of HBV-related HCC and provide theoretical basis for the treatment of HBV-related HCC.Objective:To explore the expression level of SIRT1 in HBV-related HCC,further to analyze the relationship between HBx and SIRT1,and finally to fully analyze the molecular mechanism of SIRT1/HBx regulating HBV-related HCC,so as to provide new ideas for the treatment of HBV-related HCC.Methods:1.To clarify the relationship between SIRT1 and HBV-related HCC metastasis:Tumorous tissues from 10 patient diagnosed with HBV-related HCC were collected and further divided into two groups（with or without metastasis）.Then,the mRNA and protein level of SIRT1in those tissues were detected by real-time PCR and Western blot,respectively.Meanwhile,the protein levels of epithelial-mesenchymal transition（EMT）relative markers（E-cadherin,N-cadherin and Vimentin）in those tissues were detected by Western blot.2.To explore the relationship between SIRT1 and HBV:The mRNA and protein levels of SIRT1 in HBV-expressing HCC cells were examined by real-time PCR and Western blot.3.To explore the relationship between SIRT1 and viral protein:HBx,HBc,HBs and HBp plasmids were transiently transfected into hepatoma cell line Huh-7,and the mRNA and protein levels of SIRT1 were detected by real-time PCR and Western blot;SIRT1 overexpressing and silencing in HBx induced Huh-7 and HepG2 cells,then the protein level of HBx was detected by Western blot.4.To clarify the function of SIRT1 in HBx-mediated HCC:SIRT1overexpressing and silencing in Hep AD38 and HBx induced Huh-7 and HepG2 cells.The proliferation,migration and invasion ability of cell were analyzed by Western blot,cell proliferation assay,wound healing assay and transwell assay.5.To evaluate the anti-tumor effect of SIRT1 inhibitor nicotinamide:MTT assay was used to detect the cytotoxicity of nicotinamide in Hep AD38,HepG2 and Huh-7 cells;cell proliferation experiment and wound healing experiment were used to analyze the effect of nicotinamide on the proliferation and migration of HBx induced Huh-7and HepG2 cells.Results:1.SIRT1 is closely related to the metastasis of HBV-related HCC:we found that the expression of SIRT1 were obviously increased in patients with metastasis compared to the patients without metastasis.Meanwhile,Compared with the non-metastasis group,the protein level of epithelial markers（E-cadherin）was decreased in metastasis group,while the protein level of mesenchymal markers（N-cadherin and Vimentin）were increased.2.HBV could upregulate the SIRT1 expression:compared with the controls,the mRNA and protein levels of SIRT1 were upregulated in HBV-expressing HCC cells.3.Positive interaction between SIRT1 and HBx in HCC cell lines:HBx overexpressing can increases the mRNA and protein levels of SIRT1,and SIRT1 overexpressing can increases the protein level of HBx.4.HBx promotes hepatocarcinogenesis by upregulating SIRT1:functional studies showed that SIRT1 overexpressing can significantly enhances the proliferation,migration and invasion of Hep AD38 and HBx induced HepG2 and Huh-7 cells,while SIRT1 silencing is on the contrary.5.SIRT1 inhibitor nicotinamide has antitumor effect:the CC₅₀of nicotinamide in Hep AD38,HepG2 and Huh-7 cells were 58.3m M,52.9m M and 55.7m M,respectively;nicotinamide can inhibits the proliferation and migration of hepatoma cells induced by HBx in a dose-dependent manner.Conclusions:Our findings confirmed that the expression level of SIRT1 was significantly increased in HBV-related HCC,and clarified that SIRT1 could promote the proliferation,metastasis and invasion of liver cancer mediated by HBx.