Signal Pathway of Cell Migration Mediated by HBx Figure 2. NF-kB 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 mM) 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-kB/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-kB/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-kB/p65 were examined by western blot analysis. doi:10.1371/journal.pone.0031458.g002 activation [28,30,34]. Therefore, we speculated that NF-kB may be responsible for the OPN-induced upregulation of Capn4. Luciferase reporter gene assays revealed that knockdown of NF-kB (Si-NF-kB) resulted in the abolishment of enhanced OPN- mediated promoter activity of Capn4 in a dose-dependent manner (Figure 4A). Additionally, the upregulation of Capn4 mediated by the overexpression of OPN could be attenuated by Si-NF-kB at the mRNA and protein levels in a dose-dependent manner (Figure 4B and C). Thus, we conclude that NF-kB is responsible for the OPN-mediated upregulation of Capn4. OPN is upregulated by Capn4 in a positive feedback manner Previously, we found that the tumor cell maintained proliferation and migration in a positive feedback manner [10,11,35]. Therefore, we hypothesize that Capn4 may be involved in the activation of OPN in a positive feedback manner as well. To test the hypothesis, we determined the effect of Capn4 on the regulation of OPN in HepG2-X (or H7402-X) cells by siRNA targeting Capn4 mRNA (Si-Capn4). Interesting- ly, we found that the enhanced promoter activity of OPN could PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e31458