Signal Pathway of Cell Migration Mediated by HBx 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. doi:10.1371/journal.pone.0031458.g005 OPN, Capn4 and NF-kB are involved in HBx-mediated hepatoma cell migration Furthermore, we examined the effect of OPN and Capn4 on the migration of hepatoma cells mediated by HBx. Wound healing assays showed that HepG2-X cells exhibited a much greater ability to repair the wound compared to control cells. However, the migration ability of HepG2-X cells could be suppressed by the treatment with siRNAs targeting HBx, OPN, Capn4 and NF-kB (Figure S5A, B and Figure S6A, B, see Text S1), respectively. The transient transfection of pCMV-X significantly promoted the migration of HepG2 cells, but the transient transfection of pCMV- HBc or pCMV-HBs failed to enhance the migration. In addition, the transient transfection of pcDNA-OPN and pcDNA-Capn4 also promoted the migration of HepG2 cells (Figure S5C, S5D, see Text S1), suggesting that OPN and Capn4 are involved in the promotion of HBx-mediated hepatoma cell migration. PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e31458