Effects and mechanisms of hepatitis B virus X protein on invasion and migration of hepatocellular carcinoma cells

Abstract

Objective To explore the effects and mechanisms of hepatitis B virus-X protein (HBx) on invasion and migration of hepatocellular carcinoma (HCC) cells. Methods The retrospective cohort study was conducted. The clinicopathological data of 30 patients with liver tumor (20 with HCC and 10 with benign tumor of liver) who were admitted to the Affiliated Hospital of Xuzhou Medical College between July 2014 and July 2015 were collected. HCC tissues of 20 patients with HCC (with history of HBV infection) were collected by surgical resection and peritumoral normal tissues (outside of tumor capsule) of 10 patients with benign tumor of liver (without history of HBV infection) were collected. The expressions of epidermal growth factor receptor 3 (ErbB3) in HCC tissues and peritumoral normal tissues were detected by immunohistochemistry (IHC). The relative expressions of ErbB3 and HBx in HCC tissues and peritumoral normal tissues were detected by Western blot, and relative expressions of ErbB3 in HepG2 of which green fluorescent protein (GFP) and GFP-HBx were respectively transfected were detected. The relative expressions of ErbB3 mRNA in HepG2 transfected by GFP and GFP-HBx were detected by real-time polymerase chain reaction (RT-PCR). The migration and invasion of HepG2 were respectively detected by Transwell assay with and without matrix. The measurement data with normal distribution were represented as ±s. The comparisons between groups were evaluated with the independent-sample t test. Correlation analysis was done by the Pearson test. Results (1) The expressions of ErbB3 were detected by IHC: relative value of mean optical density (MOD) of ErbB3 in HCC tissues of 20 patients with HCC and peritumoral normal tissues of 10 patients with benign tumor of liver were 2.54±1.33 and 0.99±0.29, respectively, with a statistically significant difference (t=6.542, P<0.05). (2) The relative expressions of ErbB3 and HBx were detected by Western blot: relative expressions of ErbB3 and HBx were respectively 0.79±0.13, 1.10±0.28 in HCC tissues of 10 patients with HCC and 1.07±0.17, 0 in peritumoral normal tissues of 10 patients with benign tumor of liver, with statistically significant differences (t=3.229, 19.486, P<0.05). The results of Pearson test showed that there was a positive correlation of expression between ErbB3 and HBx in HCC tissues (r=0.637, P<0.05). (3) The relative expressions and transcriptional levels of ErbB3 were detected by Western blot and RT-PCR: relative expressions of ErbB3 in HepG2 of which GFP and GFP-HBx were respectively transfected were 0.75±0.11 and 1.10±0.10, respectively, with a statistically significant difference (t=4.291, P<0.05). The relative expressions of ErbB3 mRNA in HepG2 of which GFP and GFP-HBx were respectively transfected were 0.38±0.03 and 0.94±0.07, respectively, with a statistically significant difference (t=11.703, P<0.05). (4) The effects of ErbB3 on migration and invasion of HepG2: numbers of transmenbrane cell in HepG2 of which His and His-ErbB3 were respectively transfected by Transwell assay with matrix were respectively 271±18 and 463±31, respectively, with a statistically significant difference (t=8.202, P<0.05). Numbers of transmenbrane cell in HepG2 of which His and His-ErbB3 were respectively transfected by Transwell assay without matrix were respectively 315±38 and 549±34, respectively, with a statistically significant difference (t=8.310, P<0.05). Conclusion HBx protein can promote the invasion and migration of hepatocellular carcinoma cells through up-regulating expressions of ErbB3 protein. Key words: Carcinoma, hepatocellular; Hepatitis B virus X protein; Epidermal growth factor receptor 3; Invasion; Migration