| Infection with hepatitis B virus (HBV) can cause transient and chronic liver inflammation. A long-term chronic HBV infection can lead to liver cirrhosis and development of hepatocellular carcinoma. HBV infection affects behaviors and functions of host cells, on the other hand, HBV life cycles depend on the help of host proteins. The analysis of human HBV host-virus interaction was rather difficult because HBV only infect primary hepatocytes, but not well-defined cell lines. HepG2.2.15 is well-established, constitutively HBV expressing descendent cell line of HepG2, which favors HBV replication, assembly and secretion. Despite the lack of viral initial infection steps, the HepG2.2.15 cell is an ideal model for the study of intracellular HBV life cycle and effects on host of viral infection. In the present study, the strategy of comparative cell biology and proteomic methods were utilized to systemic analysis of the differences of mutli-protein complexes and secretome between HepG2.2.15 and HepG2, with the aim to discover novel durg targets and diagnostic biomarkers for HBV infection diseases. The present study includes the following two parts:1. Comparative analysis of protein complexes of the whole cellular lysates between HepG2.2.15 and HepG2.It has been suggested that nearly all biochemical processes are performed by protein complexes, especially in virus-host interaction. BN Gel is recently developed for separation and analysis of functional protein complexes. This technique offers an advantage of separating native protein complexes without dissociating them. In this study, this method was used to characterize the different multi-protein complexes from whole cellular lysates of HepG2.2.15 and HepG2 and revealed two obvious protein complexes specially existed in HepG2.2.15 cells. The two distinct complex bands were excised and subjected to the second-dimension SDS-PAGE and proteins were sequenced by Mass spectrometry. This resulted in the identification of 25 proteins. Previous reports have shown that HSP60 activates HBV pol protein both in vitro and in vivo, down-regulation of HSP60 expression blocks HBV replication. In Duck HBV model, HSP70 and HSP90 are considered as chaperone molecules required for the activity of HBV reverse transcriptase. Several lines of evidences have suggested viral life cycle might be chaperon-dependent. In this study, antibody-based supershift assay verified the validity of the distinct protein complexes. Co-immunoprecipitaion assay showed that HSP60, HSP70 and HSP90 protein physically associate only in HepG2.2.15. SiRNA-meidated HSP70 or HSP90 down-regulation prominently inhibits HBV virus production without influencing cell proliferation and apopotosis. In addition, HSP90 inhibitor, 17-AAG, also potently reduces HBV virus secretion from HepG2.2.15. These results suggest HSP60 may be coupled with HSP70 and HSP90, forming multi-chaperone machine, involved in HBV life cycle. Such chaperones could become candidate therapeutic targets for HBV infection diseases.2. Characterization and comparative analysis of secretome from HepG2 and HepG2.2.15 cells.Blood is the main source for biomarker discovery. However, plasma protein biomarker discovery is complicated by the complexity and depth of the plasma proteome, and low relative abundance expected for specific markers. In candidate discovery, raising the relative abundance of disease associated proteins by moving "upstream" of plasma to biofluids more biologically proximal to the disease site may enhance efficiency. The proximal fluids are local sinks for proteins or peptides secreted, shed or leaked from diseased tissue. Thus, secretome of tumor cells is an ideal source of biomarkers. A proteomic strategy-SDS/PAGE-LCQ was used to analyze the secretome of HepG2 and HepG2.2.15 cells cultured with serum-free medium with an aim to discover biomarkers for HCC, especially HBV associated HCC. 149 proteins were identified with high confidence, among them, 103 proteins were identified from HepG2.2.15 secretome, and 80 proteins were from HepG2. There is obvious difference between the secretome of HepG2 and HepG2.2.15 cells in subcellular localization and function categories, which may cause by HBV infection. Some secreted proteins were biomarker candidates have been reported, indicated that secretome is feasible for cancer biomarker discovery. Serum validation indicated that fetuin may be a candidate biomarker of HCC. |
