Identification And Characterization Of The Genes Associated With Human Hepatocellular Carcinogenesis

The carcinogenesis of Hepatocellular carcinoma(HCC) are multifactorial, multifunctional and multistage. Some presume the carcinogenesis results from serial molecular changes caused by altered gene expression. So far it has been found that EBP1 ( ErbB-3 binding protein 1 ), PDZK1(PDZ domain containing 1) , PHLDAl(pleckstrin-homology-like domain family A,memberl), ANXA2 (annexin A2) and nm23-H2 (non-metastatic cells2) expressed extrordinarily in some tumor tissues, but in HCC it has not been reported. So the aim of this study is to identify HCC-related genes and investigate their function.In this study, EBP1 , PDZK1, PHLDA1, ANXA2 and nm23-H2 expressions were compared between the normal and HCC tissues with Northern bloting analysis, and nm23-H2 expression was also investigated in NIH3T3 cell line and some kinds of HCC cell lines. nm23-H2 gene was PCR amplified. We constructed the GFP (green fluorescent protein) - nm23-H2 fusion gene expression vector pEGFPCl- NM23-H2. Transfection of pEGFPCl-nm23-H2 into A293, HepG2, NIH3T3 cells were performed using lipofectin according to the manufacturer's protocol, and then the expressions and localization of NM23-H2 were detected under fluorescence microscope. After G-418 selection, the single clone was observed under fluorescence microscope, isolated using cloning rings and expanded. The transfected clones were strained using immunocytochemistry method. NM23-H2 protein was observed under laser scanning confocal microscopy. We measured cell growth by counting the survived cell by staining with trypan blue. To determine whether human nm23-H2 had oncogenic property, we examined the cell growth as colonies in soft agar and subcutaneously injected the nm23-H2-expressing NIH3T3 cells into nude mice. To measure the ability of nm23-H2 to induce focus formation, we transfected pEGFPCl-nm23-H2 and PCMV-RAS into Ratl fibroblasts, and then these cells were selected byG418.The results are as below:1. EBP1, PDZK1, ANXA2 and nm23-H2 mRNAs were differentially over expressed in tumor tissues compared with nontumor tissues. PHLDA1 was high expressed in nontumor tissues compared with tumor tissues.2. nm23-H2 mRNA was over expressed in hepatoma cell lines compared with nontumor cell lines.3. We constructed the GFP -nm23-H2 fusion gene expression vector pEGFPCl-nm23-H2. NM23-H2 protein expression is confined to mainly in cytoplasm.4. We examined the effect of ectopic expression nm23-H2 on colony formation and cell transformation. nm23-H2 decreased colony compared to vector control in NIH3T3 cells. However, individual colonies of cells transfected with nm23-H2 were larger in size and were more confluent that cells transfected with vector control. The nm23-H2 transfected cells grown in culture take on a transformed appearance, becoming irregularly elongated and losing contact inhibition.5. After G418 selection of NIH3T3 transfected by nm23-H2, we found clones and focus formation.6. The NM23-H2-expressing clones had an accelerated growth rate relative to the parental NIH3T3 or vector control cells, and overexpressing c-myc mRNA.7. The cell transfected with nm23-H2 growed as colonies in soft agar, which is not observed in the parental NIH3T3 cells or in vector control cells.8. We transiently transfected the cells with the nm23-H2 tagged at its amino terminus with the enhanced green fluorescent protein (GFP) and simultaneously performed immunohistochemistry with a mouse monoclonal antibody for nm23-H2. GFP fused to NM23-H2 protein expression is confined to mainly cytoplasm and less nuclei of the NIH3T3, HEK293T and Hep G2 cells, which is perfectively merged with immunoreactivity for NM23-H2 expression.9. After injecting the nm23-H2-expressing NIH3T3 cells into nude mice, tumor growth was observed, not in the parental or vector control cells.10. Ratl fibroblasts transfected with pEGFPCl-nm23-H2 and PCMV-RAS formed foci separately. The morphologies of they-induced foci were similar.These results suggest:1. EBP1, PDZK1, PHLDA1 > ANXA2 and nm23-H2 could be candidate markers of development and progression of HCC.2. In this study, we constructed the GFP-nm23-H2 fusion gene expression vector pEGFPCl-nm23-H2. This vector can expresses green fluorescence, which makes it easily detected in vivo and in vitro. So it is facility to study nm23-H2 further.3. nm23-H2 plays a causal role in the development of hepatocellular carcinoma.