in multiple tumor progression [33]. Therefore, we hypothesized that 5-LOX or COX-2 may be involved in the HBx-mediated upregulation of OPN. Interestingly, we showed that HBx could induce the upregulation of OPN in HepG2-X (or H7402-X) cells through 5-LOX, rather than COX-2 (Figure 1). We then examined the mechanism of HBx-mediated upregulation of 5- LOX. It has been reported that the constitutive activation of NF- kB is prevalent in liver tumor tissues [38]. NF-kB is one of important transcription factors regulated by HBx, which induces the expression of various target genes with various functions, such as, proliferation and migration [13,14,15,16]. Therefore, we speculated that NF-kB may be involved in the regulation of 5- LOX expression mediated by HBx. Our results demonstrated that the treatment with PDTC (a specific NF-kB inhibitor) and siRNAs against NF-kB abolished the HBx-mediated upregulation of 5- LOX (Figure 2), suggesting that NF-kB is responsible for the upregulation of 5-LOX. Our laboratory reported that HBx can upregulate Capn4 through NF-kB in the promotion of hepatoma cell migration [19]. Capn4 is a regulated subunit of calpains, and plays important roles in the regulation of intracellular activities, including cell spreading and migration [39], proliferation, apoptosis and differentiation [40]. Accordingly, OPN can promote tumor cell migration via the regulation of multiple signaling pathways and activation of metastasis-related gene expression. Thus, we supposed that OPN may be involved in the activation of Capn4 mediated by HBx. Our data confirmed that OPN was responsible for the upregula- tion of Capn4 in HepG2-X (or H7402-X) cells (Figure 3). Several studies have reported that OPN can promote down-stream effectors, such as metastasis-related genes, through the activation of the NF-kB pathway [28,30]. We next examined whether NF-kB was involved in the OPN-mediated upregulation of Capn4. The results showed that NF-kB was responsible for the event (Figure 4). HBx frequently regulates signal transduction in a feedback manner [35]. Then, we examined that whether Capn4 is involved in the activation of OPN. Our data showed that Capn4 was able to upregulate OPN in a positive feedback manner (Figure 5). NF-kB plays an important role in regulation of 5-LOX and OPN mediated by HBx as above. Thus, we try to demonstrate whether Capn4 regulates OPN through NF-kB as well. Our finding showed that NF-kB was also involved in the Capn4-mediated upregulation of OPN indeed (Figure 6), suggesting that NF-kB is an important factor in regulation of proteins involving cell migration. To show that the above observations are relevant to HBx expression during an HBV infection, we further examined the effect of HBx on regulation of NF-kB, 5-LOX, OPN and Capn4 in HepG2.2.15 cells using HBx RNAi. We found that HBx was able to upregulate the expression of the proteins in the cells (Figure S3). Meanwhile, we showed that the overexpression of hepatitis B virus surface antigen (HBsAg) gene or hepatitis B virus core antigen (HBcAg) gene failed to affect the promoter activities of OPN and Capn4 (Figure S4), supporting that HBx is responsible for the upregulation of NF-kB, 5-LOX, OPN and Capn4. In function, we revealed that those factors such as OPN, Capn4 and NF-kB were involved in hepatoma cell migration promoted by HBx (Figure S5, S6). Interestingly, our finding showed that a multiple factors such as NF-kB, 5-LOX, OPN and Capn4 were involved in the regulation of cell migration meditated by HBx with a cascade signaling transduction, in which NF-kB was responsible for regulation of all other three factors. Growing evidence reported that HBx, OPN and Capn4 were able to activate transcription factor NF-kB [13,28,30,36]. Importantly, OPN and Capn4 affected each other through NF-kB in a positive feedback loop. The data suggested that those factors regulate cell Signal Pathway of Cell Migration Mediated by HBx migration in a network manner. Thus, HBx drives a multiple signaling, such as, NF-kB, 5-LOX, OPN and Capn4, in positive feedback loop manner to promote hepatoma cell migration. Taken together, we conclude that HBx can enhance hepatoma cell migration through the activation of NF-kB, 5-LOX, OPN and Capn4 cascade loops with multiple cross-talk events, which contribute to the sustained promotion of cell migration mediated by HBx. Moreover, NF-kB plays an important role in the cascade loops of signaling pathways. Our finding provides new insight into the mechanism of hepatoma cell migration promoted by HBx. Materials and Methods Plasmids, reagents and siRNAs The plasmids pCMV-X, pcDNA3.0, pSilencer3.0-X, pGL3- Basic, pGL3-control, pGL3-Capn4 and renilla luciferase reporter vector pRL-TK were previously described [19,41]. The pGL3- OPN plasmid was the firefly luciferase reporter plasmid containing the full-length OPN promoter sequence [42]. The pcDNA3.0- OPN and pcDNA3.0-Capn4 plasmid were the eukaryotic expression vector containing the full-length OPN or Capn4 mRNA sequence. MK886 (an inhibitor of 5-LOX), Pyrrolidine dithiocarbamate (PDTC, an inhibitor of NF-kB) and indometh- acin (Indo, an inhibitor of COX-2) were purchased from Sigma- Aldrich (USA). The enzyme immunoassay kit used for measure- ment of leukotriene B4 (LTB4) was purchased from Adlitteram Diagnostic Laboratories (USA). The siRNAs targeting the human mRNA of OPN (targeting sequence: 59-GCCACAAGCAGTC- CAGATT-39; D28759) [43], 5-LOX (targeting sequence: 59- GCGCAAGTACTGGCTGAATGA-39; NM_000698), NF-kB/ p65 (targeting sequence: 59-ACAAGGTGCAGAAAGAGGACA- 39; NM_021975) [44], Capn4 (targeting sequence: 59- GCTTTTGTTCTCTCAGTAC-39; NM_001749) [13] and the negative control siRNA were designed and synthesized by RiboBio (Guangzhou, China). Cell culture Human hepatoma HepG2 and H7402 cells, HepG2-P/H7402- P cells (stably transfected with the empty pCMV-Tag2B vector plasmid) and HepG2-X/H7402-X cells (stably transfected with the pCMV-X plasmid) were maintained in Dulbecco’s modified Eagle’s (DMEM) medium (Gibco, USA) supplemented with heat inactivated 10% fetal calf serum (FCS), 100 U/ml penicillin, and 100 mg/ml streptomycin in a humidified atmosphere of 5% CO2 and 95% air at 37uC [35]. Construction of the human OPN and Capn4 eukaryotic expression plasmid The full-length human OPN cDNA (942 bp, Gen ID: 6696) and human Capn4 cDNA (801 bp, Gen ID: 826) were cloned into the EcoRI and XhoI sites of the eukaryotic expression vector pcDNA3.0, using human cDNA as a template, termed pcDNA3.0- OPN or pcDNA3.0-Capn4, respectively. The primers used are listed in table S1. RNA interference (RNAi) HepG2-X (or H7402-X) cells were transfected with the pSilencer-X vector that produces siRNA that targets the HBx mRNA or with control siRNA [41]. Duplex siRNAs targeting the human mRNA of OPN, 5-LOX and Capn4 were introduced into HepG2-X (or H7402-X) cells according to the manufacturer’s instructions. Duplex siRNA targeting the human NF-kB/p65 mRNA were transfected into HepG2 (or H7402) cells with overexpression of OPN according to the manufacturer’s instruc- PLoS ONE | www.plosone.org 9 February 2012 | Volume 7 | Issue 2 | e31458