Investigation Of Signaling Pathways Involved In Hepatocarcinogenesis Mediated By Hepatitis B Virus X Protein Mutants

Infection of hepatitis B virus (HBV) is closely related to the occurrence and development of hepatocellular carcinoma (HCC). HBV X protein (HBx), encoded by HBV X open reading frame (ORF), plays an important role in the development of HCC. Accordingly, it has been suggested that the multiply-sites mutation of HBx gene is universal and frequent (about 70%) in HCC tissue from patients. However, the mechanism for HBV-induced HCC remains unclear. In the present study, we examined the signal pathways of wild-type HBx protein to indicate the mechanism involving the promotion of heptoma cell growth mediated by HBx. HBx gene was amplified by HBx-specific PCR in tumor tissue samples and corresponding non-tumor tissue samples from Chinese patients with HCC to find sevel novel HBx mutants. We focused on investigating the mechanism involving the enhancement of heptoma cell growth mediated by the natural mutant of HBx truncated 27 amino acids at the COOH-terminal (termed HBx△127). This study includes three parts as follows:Part one:Investigation of the mechanism involving the promotion of cell growth mediated by HBxWe examined the effect of 5-LOX on promotion of cell growth mediated by HBx. The data showed that HBx was able to upregulate the expression of 5-LOX at levels of mRNA and protein. Enzyme-linked immunosorbent (ELISA) showed that the released leukotriene B4 (LTB4, a metabolite of 5-LOX) was increased in the conditioned media of human hepatoma HepG2-X and H7402-X cells. Moreover, we identified the mechanism involving the up-regulation of 5-LOX mediated by HBx. The data showed that the inhibition of NF-κB/p65 by PDTC (an inhibitor of NF-κB) or siRNA targeting NF-κB mRNA could attenuate the upregulation of 5-LOX mediated by HBx, suggesting that NF-κB was responsible for the upregulation of 5-LOX. Interestingly, our data showed that 5-LOX could activate NF-κB in a positive feedback manner by using MK886 (an inhibitor of 5-LOX) or siRNA targeting 5-LOX mRNA. Collectively, HBx promotes and keeps cell growth via a positive feedback involving 5-LOX and NF-κB. Part two:Identification of HBx gene mutants in cancer and non-cancerous tissues from HCC patientsIn this study, we try to find the novel HBx mutants in cancer and non-cancerous tissues from Chinese patients, and further investigate the characteristics of different type of HBx mutants. Therefore, HBx gene was amplified by HBx-specific PCR in 60 tumor tissue samples from Chinese patients with HCC and corresponding non-tumor tissue samples from the same group of HBsAg-positive patients. The data demonstrated that HBx gene was positive in 44 out of 60 tumor and non-tumor tissue samples (73.3%). We further analyzed the sequence of HBx gene, which showed that there were total 35 different types of point mutations in HBx protein from cancer tissues or non-cancerous tissues from 44 patients, respectively. Furthermore, we found that a novel phenotype of HBx gene multiple site mutation at the point of aa 30,33,38,88,144 from tumor samples and mutations at aa 31,40,87,94 from non-tumor samples were frequently detected. Importantly, the multiple site mutation at aa 30,33,38 from tumor samples and the single site mutation at aa 94 have never been reported by other study up to now.Part three:Investigation of the mechanism involving the promotion of cell growth mediated by the mutant of HBx (HBx△127)Previously, we identified a natural mutant of HBx truncated 27 amino acids at the COOH-terminal (termed HBx△127), which strongly enhanced cell growth. In the present study, we focused on investigating the mechanism. First, we established the engineered cell lines HepG2-X△127/H7402-X△127 (stably transfected with the pCMV-X△127 plasmid). Then, we examined the effect of HBxA127 on the transcription of FAS and expression level of SREBP-lc. The data showed that HBxA127 strongly increased the promoter transcriptional activities of FAS and expression level of SREBP-lc by luciferase reporter gene assays, real-time PCR, ELISA and western blotting. Furthermore, we found that MK886 (an inhibitor of 5-LOX) could abolish the upregulation of FAS and SREBP-lc in a dose-dependent manner by luciferase reporter gene assays, real time RT-PCR and western blotting, suggesting that 5-LOX is responsible for the upregulation of FAS and SREBP-1c mediated by HBxA127. Moreover, we observed that HBx△127 could upregulate 5-LOX and resulted in the increase of released leukotriene B4 (LTB4, a metabolite of 5-LOX) in the conditioned media by enzyme-linked immunosorbent assay (ELISA). The additional LTB4 could upregulate the expression of FAS in the heptoma cells as well. Interestingly, we found that FAS was able to upregulate the expression of 5-LOX in a feedback manner by using cerulenin (an inhibitor of FAS). Collectively, HBx△127 promotes cell growth via a positive feedback loop involving 5-LOX and FAS, in which the released LTB4 is involved in the upregulation of FAS.Taken together, our study provides new insight into the mechanism involving the promotion of cell growth mediated by HBx and the HBx mutant HBx△127. It is important for illustrating the molecular mechanism of HBX-related HCC, which is valuable in early diagnosis, prevention and therapy of HCC.