The Role Of Hepatitis B Virus X Gene Subtype EHBH2 In Development Of Hepatocellular Carcinoma And Clinical Significance Research

As one of the most common cancers of digestive system, Hepatocellular carcinoma (HCC) threatens people’s health severely and becomes the second cause of death worldwide. Viral hepatitis, hepatic cirrhosis, aflatoxins, pollution of drinking water and heritable fators are the main causes of HCC. In western countries, HCC is mainly caused by alcoholic cirrhosis. However, Hepatitis B Virus(HBV) is considered the major risk factors of HCC in China by triggering chronic viral B hepatitis and hepatic cirrhosis. According to the epidemiological investigations, about 100 million patients suffer from chronic viral B hepatitis every year and 5% of them may die from HBV-related disease. Therefore, to reduce the incidence and mortality rates of HCC, it is essential to deeply investigate the mechanism and effects of HBV in the development of HCC.HBV is one of hepatotropism and nonenveloped doublestranded, circular DNA virus. It could propagate in liver cells and induce inflammatory response after activation of the immune system by infection of host hepatocytes. In addition, HBV contribute to the pathogenesis and development of HCC through multiple complicated mechanisms such as integration of genetic fragments and transactivation. Hepatitis B Virus X Gene(HBx) is the smallest unstructured gene of the four open reading frames of HBV, which consists of 452-465 nucleotide and participates in HBV infections and replications directly. Accumulated studies confirmed that HBx protein could regulate numerous signals of host hepatocytes by comprehensive transactivation manners to generate many biological functions in liver cell, such as cellular apoptosis, generation cycle, cell proliferation, autophagy and migratory aptitude. Additionally, datas of whole genome sequencing show that 80% of the HBV integration fragments contains the fragement of HBx. Therefore, the instability of genomes, activation of oncogenes and inactivation of tumour suppressor genes by fragment integration of HBx might play an important role in the development of HCC.HBV DNA is more likely to mutate in the process of replication in HCC because of reverse transcription. There are 8 subtypes (A-H) and different quasispecies according to the HBV mutant degree with different HBx sequences. In addition, the HBx has 4 serotypes, including " ayw, adr, adw and ayr".Furthermore, point mutation, COOH-terminal truncation mutation, deletion mutantion is commen in the HBx sequences. These research indicate that the HBx DNA sequences have their polymorphisms. The pathogenesis and development of HCC could influenced by different HBV hypotypes and HBx mutations, which proclaim that HBx polymorphism may play a pivotal biological function in the progress of HCC. Therefore, to investigate the HBx polymorphism systematically and explore the underlying biological functions and explore the underlying biological functions and clinical manifestations of HBx in HCC might provide new sights for prevention and therapy of HCC.Part One To Analysis the HBx Genetic Polymorphisms of HBV-related HCC Patients And Its Clinical CorrelationsObjective:To detect the HBx genetic polymorphisms of HBV-related HCC patients and analysis its clinical correlations, and to find the HBx subtype which have clinical significances.Methods:We collected the tissues,serum and clinical data files of the tumor and nontumor tissues from the multicenter HBV-related HCC patients, and then we run long-time follow-up visit. Total DNA are extracted from both tumor and nontumor tissues and HBx fragments are amplifyed by nest PCR.Accurate HBx sequences are acquired by sequence. Next we used the Molecular Evolutionary Genetics Analysis 6.05 (MEGA 6.05) and Cluster 3.0 softwares to find out the different sites of HBx and make cluster analysis. We also used χ2-test, Kaplan-meier and Log-rank to analyse the clinical significance of genetic polymorphisms.Results:1) We precisely obtained 213 HBx sequences from 287 tumor tissues and 268 from 287 nontumor tissues; 2) The HBx sequences of tumor tisseus were incomplete consistent with that of nontumor tissues. The portion of the same sequences between tumor and nontumor tissues was only 39%, and the distribution of diversified sites were dispersed and irregular;3) The HBx polymorphrisms of tumor and nontumor tissues contained EHBH1, EHBH2 and EHBH3 in first cohort according to the cluster analysis; 4) The HCC patients with HBx-EHBH2 had a better prognosis and was correlated with many clinical indexes. The OS of the patients with HBx-EHBH2 were significantly higher than the other subtypes(tumor, P=0.042; nontumor, P=0.041). And the OS was correlated with the peplos of cancer tissues (P=0.002), while it was correlated with hepatic cirrhosis (P=0.000), TNM staging(P=0.033), peplos(P=0.010) and child tumor(P=0.012) for nontumor tissues; 5) The verification cohort proved that HBx-EHBH2 was correlated with patients’ prognosis. The patients with HBx-EHBH2 had a tendency of good prognosis; 6) The HBx sequences of serum was consistent with that of nontumor; 7) The HBx-EHBH2 of serum was correlated with HCC patients’prognosis and the patients with HBx-EHBH2 had a much higher OS(P=0.032).Conclusions:The HBx genetic polymorphisms exist in the tumor and nontumor of HCC patients and the HBx-EHBH2 has important for the development of HCC.Part Two Systematically Analysis the Integrations of HBx-EHBH2 in the Human GenomeObjective:To analysis the integration of HBx-EHBH2 in human genome integration,including the integral frequencies and sites.Methods:We adopted the HVID(High-throughput Viral Integration Detection) technology to test the target captured sequences of HBV inserted fragments. Then we designed the primers according to the HBV breakpoints, and checked integral sites by means of PCR and Sanger sequencing.Results:1) The integral frequencies of HBx-EHBH2 were consistent with other subtypes, including the total integral frequencies (tumor, P=0.52; nontumor, P=0.19), integral patients number (tumor, P=0.514; nontumor, P=0.228), integral frequencies of TERT (tumor, P=0.933), integral patients number of TERT(tumor, P=0.819) and the integral frequencies of exon, intron, genetic intermediate zone and promoter.2) The hot integral site genes or neighbouring genes were different from other subtypes in the both of tumor and nontumor tissues.3) The functions of integral site genes or neighbouring genes of HBx-EHBH2 differed from other subtypes.Conclusions:The human genome integral sites of HBx-EHBH2 differed from other subtypes, which might educe different biological functions.Part Three The Role and Mechanisms of HBx-EHBH2 in the HBV Related HCCObjective:To explore the biological functions and mechanisms of HBx-EHBH2, which could bring about a better prognosis.Methods:We synthesized the whole genomes of 5 type of HBx sequences, constructed plasmids, and synthesized the lentivirus. Then we transfected the hepatoma carcinoma cell and built corresponding stable transfected cell lines expressed different type of HBx. We examine the effect on cell proliferation of HBx subtypes via CCK8 and clone formation assay, and detected the generation cycle and apoptosis of cell lines through flow cytometry. Also we identified the functions in vivo of HBx subtypes by mice subcutaneous bearing cancer tests. Next, we predicted the spatial configuration of different HBx proteins by protein structure forecast software. At last, by using gene expression profile microarray,we investigate the different genes expression in subtype cell lines of HBx.Results:1) We successfully constructed stable cell lines with 5 HBx subtypes, including HBx-A, HBx-B, HBx-EHBH2, HBx-EHBH2A in two hepatoma carcinoma cell lines SMMC-7721 and Huh7; 2) CCK8 and clone formation tests also verified that HBx-EHBH2 and HBx-EHBH2A had much lower effect on cell proliferations; 3) HBx-EHBH2 and HBx-EHBH2A had the minimal effect on cell cycle which imitate to NC group; 4) HBx-EHBH2 and HBx-EHBH2A cell lines had poor depressant effects on cell apoptosis; 5) HBx-EHBH2 also had poorer actions on promoting tumor grow, which were consistent to NC group; 6) The results of protein structure forecast showed that the structure of HBx-EHBH2 differed from that of HBx-B; 7) Targeted genes of HBx-EHBH2 are different from those of HBx-B, and biological functions and pathways are various.Conclusions:HBx-EHBH2 had inferior biological functions compared to other HBx subtypes, which was exerted by HBx protein.