Hrp-3 Up-regulated Expression In Hepatocellular Carcinoma And Biological Significance

HRP-3(hepatoma-derived growth factor related protein 3) is a new member of HDGF (hepatoma-derived growth factor) family and we had got an international patent for this novel protein. In current study, we carried the study on the function of HRP-3. In normal physical condition, it expressed mainly in human testis, brain and heart, but less in other tissues. Howerer, its expression level was higher in hepatocellular carcinoma (HCC) and hepatoma cells. To discuss the significance of HRP-3 in physiological and pathological process in HCC, we proved its expression level was up-regulated in hepatoma cells especially in the vessels or vessel region in HCC. Then we applied the transient expression in mammalian cells and bacteria expression to examine whether HRP-3 could be secreted from cells like HDGF protein. The results showed that HA-HRP-3 and GST-HRP-3 fusion protein could be detected in the concentrated culture medium using the anti-HA or anti-GST monoclonal antibody. Moreover, the subcelluar location showed that enhanced green fluorescent protein (EGFP)-tagged HRP-3 fusion protein was in nucleus. These results suggested that HRP-3 had the characteristic as secreted protein and nuclear protein.To find the effect of HRP-3 as a secreted protein, we recombinanted the HRP-3 protein and used it as a supplement in the foundamental cell culture medium. The results displayed that when the concentration of HRP-3 was within 50-1000ng/mL, it could promote the proliferation of NIH3T3, HUVEC and HepG2 cells. HRP-3 had the most distinct effect on HUVECs, followed by NIH3T3 cells. HRP-3 could also stimulate the growth of HepG2 cells. It implied that the increased HRP-3 expression in HCC may potentially related with the growth of vessels in tumor and hepatoma cells during the pathogenesis of hepatocellular carcinoma.According to these studies, we investigated the mechanism on cells proliferation induced by HRP-3. Adding HRP-3 protein by the concentration of 50-1000ng/mL to NIH3T3 cells, the phosphorylation of MEK and ERK could be rapidly, transiently and specially enhanced in 5-10min, and this activation could be inhibited by the MEKinhibitor PD98059. But the P38 and JNK signal pathway were not influenced by HRP-3. To confirm the physiological effect of the kinase activation, we used the dual-luciferase reporter system to check the response of MAPK downstream signal elements, the results showed that HRP-3 protein could enhance E-box and SRE (DNA responsone elements) dependent transcription in l-4h. These finding suggested that HRP-3 up-regulated expression in HCC stimulated the tumor growth by increasing its intracellular and extracellular expression to strengthen the MAPK signal in hepatoma cells and vascular endothelial cells.In addition, we found the growth rate was higher and the MAPK signal was enhanced in the cell lines stablly expressed the exogenous HRP-3. We deduced it may induced by the increased HRP-3 expression in the culture medium, but whether it had the effect in the nucleus needs more study in future.Moreover, we had taken another simple study on cloning and characterize of a novel human cellular retinaldehyde binding protein-like (CRALBPL) gene. CRALBPL is a new member of a widespread lipid binding SEC14-like protein family with CRAL-TRIO domain, which was predicted and subsequently isolated from the human adult brain cDNA library. The CRALBPL gene was mapped to human chromosome 8q12.2, consisted of 1694 bp and had a open reading frame in length encoding a putative 354 amino acids with a CRAL-TRIO domain in 118-279 aa. The result of RT-PCR amplification in 18 human tissues indicated that CRALBPL gene was mainly expressed in brain. The alignment of CRAL-TRIO domain showed that CRALBPL had 45% identity with human CRALBP. Subcellular location revealed that CRALBPL protein was located in the cytoplasm of Hela cells. Western blotting indicated that the CRALBPL had a molecular weight of about 40kD. For CRALBP playing a role in the vertebrate visual process as a substrate-routing protein, it carries 11-cis-retinol and 11-cis-retinaldehyde as endogenous ligands and may be a functional component of the visual cycle. The clone of novel CRALBPL gene will develop a new and broad field to understand visual process.