Hepatitis B virus X protein promotes epithelial-mesenchymal transition of hepatocellular carcinoma cells by regulating SOCS1

Abstract

Hepatocellular carcinoma (HCC), a primary type of liver cancer, is one of the leading causes of cancer related deaths worldwide. HCC patients have poor prognosis due to intrahepatic and extrahepatic metastasis. Hepatitis B virus (HBV) infection is one of the major causes of various liver diseases including HCC. Among HBV gene products, HBV X protein (HBx) plays an important role in the development and metastasis of HCC. However, the mechanism of HCC metastasis induced by HBx has not been elucidated yet. In this study, for the first time, we report that HBx interacts with the suppressor of cytokine signaling 1 (SOCS1) which negatively controls NF-κB by degrading p65, a subunit of NF-κB. NF-κB activates the transcription of factors associated with epithelial-mesenchymal transition (EMT), a crucial cellular process associated with invasiveness and migration of cancer cells. Here, we report that HBx physically binds to SOCS1, subsequently prevents the ubiquitination of p65, activates the transcription of EMT transcription factors and enhance cell migration and invasiveness, suggesting a new mechanism of HBV-associated HCC metastasis. [BMB Reports 2022; 55(5): 220-225].

Fig. 1

HBx upregulates the level of p65. (A) HepG2.2.15 cells were transfected with shRNA against HBx. At 72 h after transfection, levels of HBx, p65, IκBα, IKK, and phosphorylated IKK were determined by immunoblotting with anti-HBx, anti-p65, anti-IκBα, anti-IKK, and anti-phosphorylated IKK antibodies. (B) Hep3B cells were transfected with HBx shRNA and scramble shRNA. At 72 h after transfection, cell lysates were analyzed by Western blotting with anti-HBx, anti-p65, anti-IκBα, anti-IKK, and anti-phosphorylated IKK antibodies. (C) HepG2 cells were transiently transfected with various amounts of Flag-HBx plasmid. At 48 h after transfection, cell lysates were prepared and analyzed by Western blotting with anti-Flag, anti-p65, and anti-β-actin antibodies.

Fig. 2

HBx inhibits SOCS1-mediated suppression of p65 and physically binds to SOCS1. (A) HepG2 cells were co-transfected with HA-p65, Flag-HBx, and Myc-SOCS1 plasmids. At 48 h after transfection, NF-κB activities were determined by a dual luciferase reporter assay. Results are denoted as relative luciferase activities. Data shown are means ± SD of values from three independent experiments. (B) HepG2 cells were co-transfected with Flag-HBx and Myc-SOCS1 plasmids. Cell lysates were subjected to Western blot analysis using antibodies against Flag, p65, Myc, and β-actin. (C) HepG2 cells and Hep3B cells were transfected with SOCS1 shRNA and scramble shRNA. At 72 h after transection, cell lysates were prepared and analyzed by Western blotting with anti-SOCS1, anti-p65, anti-IKK antibodies. (D) L ysates of Hep3B cells expressing HBx constitutively were prepared and immunoprecipitated with anti-HBx antibody. Immunoprecipitated complex was analyzed by Western blotting with anti-HBx and anti-SOCS1 antibodies. (E) 293T cells were transfected with Flag-HBx and Myc-SOCS1 plasmids. Cell lysates were immunoprecipitated with anti-Myc and analyzed by Western blotting with anti-Myc and anti-Flag antibodies.

Fig. 3

HBx stabilizes p65 by blocking p65 ubiquitination induced by SOCS1. (A) HepG2 cells were co-transfected with Flag-HBx and Myc-SOCS1 plasmids. At 48 h after transfection, cells were treated with 400 μM cycloheximide for 0, 12, and 24 h. Cell extracts were analyzed by Western blotting with anti-Flag, anti-Myc, anti-p65, and anti-β-actin antibodies. (B) Hep3B cells were transfected with Myc-SOCS1 plasmid and HBx-shRNA plasmid. Cell extracts were prepared and analyzed by Western blotting with anti-p65, anti-HBx, anti-Myc, and anti-β-actin antibodies. (C) HepG2 cells were co-transfected with Flag-HBx, Myc-SOCS1, and GST-p65 plasmids. At 48 h after transfection, cells were treated with 20 μM MG-132 for 4 h. Cell extracts were prepared, pulled down with Glutathione-Sepharose 4B, and analyzed by Western blotting with anti-Myc and anti-GST antibodies. (D) HepG2 cells were co-transfected with Flag-HBx, Myc-SOCS1, and His-p65 plasmids. At 48 h after transfection, cells were treated with 20 μM MG-132 for 4 h. Cell extracts were immunoprecipitated with anti-Myc antibody and analyzed by Western blotting. (E) HepG2 cells were co-transfected with Flag-HBx, Myc-SOCS1, GST-p65, and HA-Ub plasmids. At 48 h after transfection, cells were treated with 20 μM MG-132 for 4 h. Cell extracts were pulled down with Glutathione-Sepharose 4B and analyzed by Western blotting.

Fig. 4

HBx promotes cellular migration and invasion by restoring levels of EMT factors suppressed by SOCS1. (A) HepG2 cells were transfected with Flag-HBx and Myc-SOCS1 plasmids. At 48 h after transfection, cell lysates were analyzed by Western blotting with anti-Flag, anti-p65, anti-Twist1, anti-Snail, and anti-β-actin antibodies. (B) HepG2 cells were transfected with Flag-HBx and Myc-SOCS1 plasmids. Cell lysates were prepared and analyzed by Western blotting with anti-Flag, anti-p65, anti-vimentin, anti-E-cadherin, and anti-β-actin antibodies. (C) HepG2 cells were transfected with Flag-HBx and Myc-SOCS1 plasmids. At 48 h after transfection, shapes of transfected HepG2 cells were analyzed by light microscopy. Scale bar = 50 μm. (D) Wound healing assay was carried out using HepG2 cells transfected with Flag-HBx and Myc-SOCS1 plasmids. Representative images were taken at 24 h after scratching. (E) HepG2 cells were transfected with Flag-HBx and Myc-SOCS1 plasmids. The invasion ability of cells was measured using Transwell cell culture chambers.