Signal Pathway of Cell Migration Mediated by HBx 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. doi:10.1371/journal.pone.0031458.g003 be abolished by Si-Capn4 in a dose-dependent manner (Figure 5A). Additionally, the expression of OPN at the levels of mRNA and protein could be attenuated by Si-Capn4 in a dose-dependent manner (Figure 5B and C). The data suggest that OPN is regulated by Capn4 in a positive feedback manner in HepG2-X (or H7402-X) cells. To further confirm that Capn4 is able to upregulate OPN, we examined the effect of Capn4 on the expression of OPN by transient transfection of pcDNA3.0- Capn4 in HepG2 cells. Luciferase reporter gene assays showed that overexpression of Capn4 led to a strong enhancement of promoter activity of OPN in a dose-dependent manner in HepG2 cells (Figure 5D). Furthermore, RT-PCR and western blot confirmed that the expression levels of OPN were increased by the overexpression of Capn4 in a dose-dependent manner (Figure 5E and F). Our data suggest that Capn4 is able to stimulate the expression of OPN. NF-kB is responsible for the Capn4-mediated upregulation of OPN It has been reported that Capn4 activates NF-kB in HeLa cells, which is involved in the survival pathway [36]. Therefore, we speculated that NF-kB may be involved in the Capn4-induced upregulation of OPN. Luciferase reporter gene assays revealed that the knockdown of NF-kB (Si-NF-kB) resulted in the PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e31458