correlated with overexpression of calpain small subunit 1 (Capn4) [17], which belongs to the calpain system [18]. Recently, we have reported that HBx could promote hepatoma cell migration through the upregulation of Capn4 [19]. Several reports have revealed that osteopontin (OPN) plays important roles in tumor cell adhesion, migration, invasion and angiogenesis [20,21,22,23]. An elevated level of plasma OPN is significantly related to cancer invasiveness and has a significant impact on tumor development and patient survival rate [24]. OPN is overexpressed in multiple tumor tissues and is associated with invasion, progression or metastasis in numerous human cancers, such as liver [25], breast and colon [26] cancer. OPN promotes tumor cell migration via the regulation of multiple signaling pathways and activation of metastasis-related gene expression. Some downstream effectors of OPN, including PI3K/Akt, EGFR, HGFR, MMPs, and NF-kB, mediate critical metastatic processes [27,28,29,30,31]. Therefore, we hypothesize that 5-LOX, NF-kB/p65 and OPN may be involved in cell migration mediated by HBx and Capn4. In the present study, we investigated the signal pathways involving hepatoma cell migration promoted by HBx. Our finding shows that HBx drives multiple cross-talk cascade loops to promote hepatoma cell migration, providing new insight into the mechanism of development of HBx-mediated HCC. Results HBx upregulates the expression of OPN in hepatoma cells To investigate whether HBx upregulates the expression of OPN, we examined the effect of HBx on the promoter activity of OPN. Our data showed that HBx could significantly enhance the promoter activity of OPN by luciferase reporter gene assay in a dose dependent manner (Figure S1A, see Text S1). Meanwhile, RNA interference (RNAi) targeting HBx mRNA mediated by pSilencer3.0-X (pSi-HBx) could abolish the increase of OPN promoter activity in a dose-dependent manner (Figure S1A, see Text S1). In addition, western blot analysis showed that HBx upregulated the expression of OPN in HepG2-X (or H7402-X) cells, respectively (Figure S1B, see Text S1). The knock down of HBx could attenuate the upregulation of OPN in the cells by pSi- HBx in a dose-dependent manner (Figure S1B, see Text S1). Thus, we conclude that HBx is able to upregulate the expression of OPN. 5-LOX is responsible for the upregulation of OPN mediated by HBx 5-LOX, a catalyzing enzyme of arachidonic acid, was overexpressed during multistage tumor progression in many neoplastic disorders including lung, breast, pancreatic cancers and HCC HBx-integrated host hepatocytes [32,33]. Our previous study found that HBx could upregulate 5-LOX and COX-2, which were involved in proliferation and migration [10,11]. Therefore, we hypothesize that 5-LOX or COX-2 may be involved in the HBx-mediated OPN upregulation. To answer this question, we treated HepG2-X cells with MK886 (an inhibitor of 5-LOX) and Indo (an inhibitor of COX-2) for 6 h. Luciferase reporter gene assays demonstrated that MK886 could abolish the enhanced promoter activity of OPN in HepG2-X cells in a dose- dependent manner (Figure 1A). However, the treatment with Indo failed to affect the promoter activity of OPN (Figure 1A). In addition, we found that 5-LOX was also able to upregulate the promote activity of OPN in HepG2 cells, but the luciferase activity was lower than that in HepG2-X cells (Figure 1A), suggesting that HBx upregulates OPN through 5-LOX. Furthermore, we found Signal Pathway of Cell Migration Mediated by HBx that siRNA against 5-LOX (Si-5-LOX) could abolish the increased promoter activity and upregulated expression of OPN protein in HepG2-X (or H7402-X) cells in a dose-dependent manner by luciferase reporter gene assay (Figure 1B) and western blot analysis (Figure 1C). Thus, we conclude that HBx is able to upregulate the expression of OPN through 5-LOX. Next, we examined the effect of HBx on the regulation of 5- LOX. We confirmed that the 5-LOX expression levels were markedly upregulated in HepG2-X (H7402-X) cells relative to the HepG2 (H7402) cells by real-time PCR and western blot (Figure S2A, S2B, see Text S1), which is consistent with our previous study [11]. HBx RNAi could abolish the upregulation of 5-LOX (Figure S2A, S2B, see Text S1). Furthermore, the data showed that the amount of LTB4, a metabolite of 5-LOX, was higher in the conditioned media of HepG2-X (or H7402-X) cells than that in HepG2 (or H7402) cells, which could be abolished by RNAi targeting HBx mRNA (Figure S2C, D, see Text S1). NF-kB is responsible for the upregulation of 5-LOX mediated by HBx Our previous study reported that HBx activates NF-kB/p65 in HepG2-X and H7402-X cell lines [19]. Moreover, we found that 60 mM PDTC (an inhibitor of NF-kB) could significantly downregulate the expression of 5-LOX mRNA and protein in HepG2-X (or H7402-X) cells (Figure 2A and B). In addition, 60 mM PDTC could decrease the amount of released LTB4 in the conditioned media of HepG2-X (or H7402-X) cells and cell lysates (Figure 2C). Meanwhile, 100 nM siRNA against NF-kB p65 was able to attenuate the levels of mRNA and protein of 5- LOX in HepG2-X (or H7402-X) cells (Figure 2D and E), which was consistent with the above data. In the control, immunoblot analysis showed that 60 mM PDTC (or 100 nM NF-kB p65 siRNA) could successfully downregulate the expression of NF-kB/p65 in nuclear extracts from HepG2-X (or H7402-X) cells (Figure 2B and E). Thus, our data suggest that NF-kB is responsible for the HBx-mediated upregulation of 5-LOX. OPN is responsible for the HBx-mediated upregulation of Capn4 Recently, we have reported that HBx upregulates the expression of Capn4 through NF-kB to promote cell migration [19]. Previous studies demonstrated that OPN was significantly associated with tumor metastasis by activating other metastasis-related genes. Therefore, we hypothesize that OPN may be correlated with the HBx-mediated upregulation of Capn4. To address this question, we examined the effect of OPN knockdown (Si-OPN) on the promoter activity of Capn4 in HepG2-X (or H7402-X) cells by luciferase reporter gene assay. The results showed that Si-OPN could abolish the enhanced promoter activity of Capn4 in a dose- dependent manner (Figure 3A). Furthermore, we detected an effect of Si-OPN on the expression of Capn4 at the mRNA and protein level in HepG2-X (or H7402-X) cells by RT-PCR and western blot analysis. The data revealed that Si-OPN could significantly attenuate the HBx-mediated upregulation of Capn4 at the mRNA and protein levels in a dose-dependent manner (Figure 3B and C). This suggests that OPN is responsible for the HBx-mediated upregulation of Capn4. To further confirm the role of OPN in the upregulation of Capn4, we examined the effect of OPN on the expression of Capn4 by transient transfection of pcDNA3.0-OPN in HepG2 (or H7402) cells. Luciferase reporter gene assays showed that the overexpression of OPN led to a strong enhancement of promoter activity of Capn4 in a dose-dependent PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e31458