Defining the Oncogenic Functions of Hepatitis B Virus-Human Fusion Transcripts in Hepatocellular Carcinoma

Chronic hepatitis B (HBV) infection IS a major etiologic factor in the development of human hepatocellular carcinoma (HCC). It is long recognized that viral integration into the host HCC genome can be detected in >85% cases, yet the inertional mutagenesis remains largely unclear. In an attempt to define viral integrants, our lab has previously defined recurring HBx/8p 11 integration site using the technique of restriction site-polymerase chain reaction (RS-PCR). This study was elaborated by cloning the full-length HBx/8p 11 chimeric transcript by 5' and 3' Rapid Amplification of cDNA Ends (RACE). Investigations conducted in this thesis revealed that this novel viral-human HBx/8pll fusion transcript functions as long non-coding RNA (ncRNA). It regulates the epithelial-to-mesenchymal transition (EMT) and triggers Wnt signalling pathway, all of which are considered important processes in the liver oncogenesis.;The full-length HBx/8p11 chimeric transcript of 674bp contains a microhomology sequence of GTG shared at the flanking juncture. Expression of HBx/8p11 fusion transcript can be detected in 23.3% of HBV-positive HCC tumors (21 out of 90 cases), where a significant correlation with shorter patient survival ( P = 0.015) and cox prognostic indicative value (P = 0.014) could be drawn. Functional knockdown of HBx/8pll resulted in marked inhibition on cell migration (P = 0.04) and invasion (P = 0.04), with upregulation of epithelial markers E-cadherin and γ-catenin expressions, and downregulation of mesenchymal marker fibronectin, suggesting its likely role in EMT phenotype. A corresponding diminution of β-catenin nuclear localization and reduced β-catenin trans activating activity (P = 0.0076) were also revealed, which enforced a role for HBx/8p11 in the membrane-bound ECadherin/β-catenin complex. Conversely, ectopic expression of full-length HBx/8p11 in immortalized hepatocyte L02 promoted cell invasiveness (P = 0.005) and migration ( P = 0.028) via promoting EMT. This effect was not observed in L02 transfected with empty vector, HBx component or flanking 8p11 sequence. GFP-tagged constructs further suggested protein encoded from HBx/8p11 was similar to the flanking HBx component, suggesting the functional phenotype observed on HBx/8p11 was likely attributed to a non-protein coding RNA transcript (ncRNA). Ectopic expression of full-length HBx/8p11 in L02 also induced colony growth. We further demonstrated in liver-specific transgenic model that mice with HBx/8p11 trans gene were more susceptible to DEN-induced tumor formation than non-transgenic littermate. These data highlights the importance for the HBx/8p11 ncRNA transcript in conferring oncogenic advantages, and may represent an elemental predisposing factor in the initiation and progression of HCC.;During the course of RACE cloning for HBx/8p11 full-length sesquence, another viral-human transcript HBx/22q11 (477bp) was also identified. Similar to the HBx/8p11 characterizations of HBx/22q11 were also carried out. HBx/22q11 expressed in 24.9% HBV-positive HCC cases (12 out of 49 cases). However, HBx/22q11 expressions do not correlate with patients' survival and clinicopathological parameters of these patients. Functional studies also revealed insignificant effect on cell migration, invasion and cell growth.;In summary, novel recurrent viral integration sites leading to the expression fusion transcripts were identified. In particular, HBx/8p11 functions as long ncRNA to promote cell motility, cell proliferation and tumor growth. Taken together with its high prevalence and significant effect in overall survival, HBx/8p11 fusion transcript is a possible molecular target for the development of successful therapeutic interventions in HCC.