Hepatitis B virus X protein drives multiple cross-talk cascade loops involving NF-κB, 5-LOX, OPN and Capn4 to promote cell migration

Abstract

Hepatitis B virus X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). However, the mechanism remains unclear. Recently, we have reported that HBx promotes hepatoma cell migration through the upregulation of calpain small subunit 1 (Capn4). In addition, several reports have revealed that osteopontin (OPN) plays important roles in tumor cell migration. In this study, we investigated the signaling pathways involving the promotion of cell migration mediated by HBx. We report that HBx stimulates several factors in a network manner to promote hepatoma cell migration. We showed that HBx was able to upregulate the expression of osteopontin (OPN) through 5-lipoxygenase (5-LOX) in HepG2-X/H7402-X (stable HBx-transfected cells) cells. Furthermore, we identified that HBx could increase the expression of 5-LOX through nuclear factor-κB (NF-κB). We also found that OPN could upregulate Capn4 through NF-κB. Interestingly, we showed that Capn4 was able to upregulate OPN through NF-κB in a positive feedback manner, suggesting that the OPN and Capn4 proteins involving cell migration affect each other in a network through NF-κB. Importantly, NF-κB plays a crucial role in the regulation of 5-LOX, OPN and Capn4. Thus, we conclude that HBx drives multiple cross-talk cascade loops involving NF-κB, 5-LOX, OPN and Capn4 to promote cell migration. This finding provides new insight into the mechanism involving the promotion of cell migration by HBx.

Figure 1. 5-LOX is responsible for the upregulation of OPN mediated by HBx.

(A) The promoter activities of OPN were detected by luciferase reporter gene assay in HepG2-X and HepG2 cells treated with MK886 or Indo, respectively (**P<0.01, ns, not significant, Student's t test). (B) The promoter activity of OPN was detected by luciferase reporter gene assay in HepG2-X (or H7402-X) cells treated with the indicated doses of siRNA targeting 5-LOX mRNA (Si-5-LOX) (**P<0.01, Student's t test). (C) The protein levels of OPN, 5-LOX and HBx were examined in the cells by western blot analysis.

Figure 2. NF-κB is responsible for the upregulation of 5-LOX.

(A, B and C) HepG2-X (or H7402-X) cells were treated with PDTC (0, 20, 40 and 60 µM) for 6 h. (A) The mRNA levels of 5-LOX were examined by real-time PCR (*P<0.05, ***P<0.001, Student's t test). (B) The protein levels of 5-LOX and NF-κB/p65 were examined by western blotting. (C) The amount of LTB4 was detected by ELISA in conditioned media or in cell lysates (**P<0.01, Student's t test). (D and E) HepG2-X (or H7402-X) cells were transfected with siRNA targeting NF-κB/p65 mRNA or control siRNAs for 48 h. (D) The mRNA levels of 5-LOX were measured by real-time PCR (***P<0.001, Student's t test). (E) The protein levels of 5-LOX and NF-κB/p65 were examined by western blot analysis.

Figure 3. OPN is responsible for the HBx-mediated upregulation of Capn4.

(A, B and C) HepG2-X (or H7402-X) cells were transfected with the indicated doses of siRNA targeting OPN mRNA (Si-OPN) and control siRNA for 48 h. (A) The promoter activity of Capn4 was detected by luciferase reporter gene assay (**P<0.01, *P<0.05 Student's t test). (B) The mRNA levels of Capn4 were detected by RT-PCR. (C) The protein levels of Capn4 were examined by western blot analysis. (D, E and F) HepG2 (or H7402) cells were transiently transfected with the indicated doses of pcDNA3.0-OPN for 48 h. (D) The promoter activity of Capn4 was measured by luciferase reporter gene assay (*P<0.05 Student's t test). (E, F) The mRNA and protein levels of Capn4 were examined by RT-PCR and western blot analysis, respectively.

Figure 4. NF-κB is responsible for the OPN-mediated upregulation of Capn4.

(A, B and C) OPN-overexpressed HepG2 (or H7402) cells were treated with the indicated doses of siRNA targeting NF-κB mRNA (Si-NF-κB) for 48 h. (A) The promoter activity of Capn4 was examined by reporter gene assay (**P<0.01 Student's t test). (B) The mRNA levels of Capn4, OPN and NF-κB/p65 were detected by RT-PCR. (C) The protein levels of Capn4, OPN and NF-κB/p65 were measured by western blot analysis.

Figure 5. Capn4 can regulate OPN in a positive feedback manner.

(A, B and C) HepG2-X (or H7402-X) cells were transfected for 48 h with the indicated doses of siRNA targeting Capn4 mRNA (Si-Capn4). (A) The promoter activity of OPN was measured by luciferase reporter gene assay (*P<0.05, Student's t test). (B, C) The mRNA and protein levels of OPN were detected by RT-PCR and western blot analysis, respectively. (D, E and F) HepG2 cells were transiently transfected with the indicated doses of pcDNA3.0-Capn4 for 48 h. (D) The promoter activity of OPN was measured by luciferase reporter gene assay (*P<0.05 Student's t test). (E, F) The mRNA and protein levels of OPN were examined by RT-PCR and western blot analysis, respectively.

Figure 6. NF-κB is responsible for the Capn4-mediated upregulation of OPN.

(A, B and C) Capn4-overexpressed HepG2 (or H7402) cells were treated with the indicated doses of siRNA targeting NF-κB mRNA (Si-NF-κB) for 48 h. (A) The promoter activity of OPN was examined by luciferase reporter gene assay (**P<0.01 Student's t test). (B) The mRNA levels of OPN, Capn4 and NF-κB/p65 were detected by RT-PCR. (C) The protein levels of OPN, Capn4 and NF-κB/p65 were measured by western blot analysis.