Investigation Of Relationships Between Hepatitis B Virus X Gene Integration/X Protein And Hepatocarcinogenesis

Infection of hepatitis B virus (HBV) is closely related to the development of hepatocellular carcinoma (HCC). The integration of HBV DNA into the host genome and the trans-activation of HBx protein play crucial roles in the development of HCC. However, the molecular mechanisms underlying HBV-induced HCC remain to be elucidated. Most researches related to HBx focused on its trans-activation role in the cells, but the role of HBV DNA integration in hepatocarcinogenesis was ignored. It has been reported that HBV DNA, especially X gene, can randomly integrate into the host genome with a very high integration rate of more than 80% in HBV-related HCC. Previously, we reported that human immortalized liver L-O2 cells could be transformed by HBx in a model of stably HBx gene-transfection cells. Therefore, we supposed that the integration of HBx gene may be directly involved in hepatocarcinogenesis. Meanwhile, HBx protein also plays an important role in the metastasis of HCC. Accordingly, osteopontin (OPN) and calpain small subunit 1 (Capn4) are related to tumor cell adhesion, migration and invasion. Therefore, we supposed that OPN and Capn4 may be involved in the promotion of hepatoma cell migration mediated by HBx. In the present study, we took advantage of cell models and clinical HCC specimens to investigate the direct role of the HBx gene-integration in hepatocarcinogenesis and the mechanism of hepatoma cell migration promoted by HBx protein. This study includes two parts as follows:Part I:Investigation of relationship between HBx gene integration and hepatocarcinogenesisEstablishment of cell models stably transfected with HBx mutantWe previously reported that the L-O2 cells stably transfected with full-length HBx gene (L-O2-X cells) could be transformed, suggesting that HBx integration may be involved in hepatocarcinogenesis. However, there were the possibilities of trans-activation by HBx proteins in the transformation mediated by full-length HBx-integration. To investigate the direct role of HBx integration in the transformation, we tried to establish cell models involving HBx-mutant gene integration, in which the mutant HBx protein lost the trans-activation function. Firstly, we randomly divided the full-length HBx gene into five fragments, which were subcloned into the pCMVTag-2B vector. The constructs were termed as pCMV-X1, pCMV-X2, pCMV-X3, pCMV-X4 and pCMV-X5, respectively. We observed that the overexpression of the 5 fragments of HBx gene failed to affect the promoter activities of NF-κB and human telomerase reverse transcriptase (hTERT) relative to wild type HBx protein in the transiently HBx mutant-transfected L-O2 cells, suggesting that the mutant HBx proteins lost the trans-activation roles. The constructed plasmids were transfected into L-O2 (or H7402) cells and selected by G418 for two weeks. The engineered cells were termed as L-02-X1,L-O2-X2, L-O2-X3, L-O2-X4 and L-O2-X5 (or H7402-X1, H7402-X2, H7402-X3, H7402-X4 and H7402-X5), respectively.Effect of HBx gene integration on the transformation of L-O2 cellsWe observed the malignant phenotype of above HBx-fragments engineered L-O2 cells. The results showed that alpha-fetoprotein (AFP) was detectable in the L-O2-X3 and L-O2-X5 cells, suggesting that L-O2-X3 and L-O2-X5 cells become HCC cells. Moreover, the two cell lines generated more clones in soft agar. Animal transplantation showed that nude mice injected with L-O2-X3 and L-O2-X5 cells gave rise to tumors after transplantation, while the others failed to form any visible tumors. This finding suggests that L-O2-X3 and L-O2-X5 cells are transformed and that HBx gene-integration is directly involved in the transformation of L-O2 cells.The mechanism of HBx gene integration-meditated transformationWe found that HBx-integration was involved in the transformation of L-O2 cells, and then we tried to identify the HBx-integration manner by HBx-Alu PCR approach in the L-02-X cells. We randomly picked up one band from the positive PCR products. The sequencing data revealed that HBx gene inserted into the upstream of a 20 bp Alu core sequence with a secondary deletion of 382-465 bp at the 3'end of HBx gene, followed by subtelomeric DNA, suggesting that HBx gene-integration results in a recombination of HBx/Alu core sequence/subtelomeric DNA in the host genome. Accordingly, we designed the PCR primers to amplify the sequence from the integrated HBx to subtelomeric DNA, to confirm the recombination in H7402-X (human hepatoma H7402 cells stably transfected HBx gene),3T3-X (NIH3T3 cell line stably transfected HBx gene), HepG2.2.15 and HepG2-X (human hepatoma HepG2 cells stably transfected HBx gene). The same recombination could be only detected in H7402-X cells.To investigate whether the recombination involved the transformation of L-O2 cells, we examined the recombination in above ten engineered cell lines stably transfected with five HBx fragments by PCR using the primers from each of the five fragments to subtelomeric DNA. Interestingly, we found that the recombination was found in the transformed L-O2-X3 and L-O2-X5 (or H7402-X3 and H7402-X5) cells but not in the non-transformed cells. The corresponding recombination just matched the L-O2-X3 and L-O2-X5 cell lines which were transformed, suggesting that the recombination of HBx gene/Alu core sequence/subtelomeric DNA was involved in the transformation. Furthermore, we provided more evidence of the transformation mediated by HBx-integraiton at the molecular level. The promoter activities of NF-κB, activator protein-1 (AP-1) and hTERT were increased in the engineered cells with the HBx integration-meditated recombination (such as, L-O2-X, L-O2-X3, L-O2-X5, H7402-X, H7402-X3 and H7402-X5). The proteins involved in proliferation and transformation, such as c-Myc, proliferating cell nuclear antigen (PCNA) and Bcl-2 were remarkably upregulated in the corresponding cells as well. However, the cell lines without the HBx meditated-recombination had no the phenotype. These results indicate that HBx integration-meditated recombination is one of crucial causes of transformation of hepatocytes.To examine the recombination in human HBV-related HCC tissues, we first detected the HBx gene in 60 cases of HCC tissues and their non-tumor ones by PCR. The positive rates of HBx gene were 73.3%(44/60) in HCC tissues and 73.3%(44/60) in their non-tumor tissues. Then, we examined the recombination of HBx/Alu core sequence/subtelomeric DNA in the above HBx-positive tissues by PCR using the primers from HBx to subtelomeric DNA. The recombination was detectable in 5 out of 44 cases HCC tissues, while in their non-tumor tissues the recombination was negative. In summary, our finding first provides evidence that the HBx integration is directly related to the hepatocarcinogenesis involving a recombination of HBx/Alu core sequence/subtelomeric DNA. It suggests that the prevention of HCC is very important.Part II:The mechanism of hepatoma cell migration promoted by HBx proteinInvestigation of the signal pathways involved in hepatoma cell migration promoted by HBx proteinRecently, we reported that HBx protein could upregulate the expression of Capn4 in the hepatoma cells. In this study, we revealed that HBx could upregulate OPN through 5-lipoxygenase (5-LOX) in hepatoma HepG2 (or H7402) cells. Moreover, OPN could upregulate the expression of Capn4 through NF-κB in the cells. Interestingly, Capn4 could regulate the expression of OPN in a positive feedback manner. In function, wound healing assay showed that OPN and Capn4 were involved in the promotion of hepatoma cell migration mediated by HBx. Thus, we conclude that HBx promotes hepatoma cell migration via a positive feedback loop involving HBx/5-LOX/OPN/NF-KB/Capn4/OPN.The mechanism of hepatoma cell migration promoted by HBx protein mutant (HBxΔ127)We previously identified a natural mutant of the HBx gene, termed HBxΔ127, which was able to promote cell proliferation. In the present study, we investigated the role of HBxΔ127 in hepatoma cell migration. The results display that HBxΔ127 strongly promotes hepatoma cell migration via activating OPN involving 5-LOX relative to wild type HBx protein.Taken together, our findings provide new insight into the mechanism of hepatoma cell migration mediated by HBx protein. Potentially, OPN and Capn4 may serve as key therapeutic targets.