The Roles Of HPO In Invasiveness Of Hepatoma Cell

Hepatopoietin(HPO) is a novel human hepatotrophic growth factor. It can specifically stimulate the proliferation of hepatocytes, and has closely relationships with the recovery of damaged liver.HPO plays prominant roles in the regeneration and protection of liver. It can stimulate the proliferation of hepatocytes, promote the recovery of damaged hepatocytes and rescue acute hepatic failure. Recent studies have indicated that an autocrine loop of HPO/HPO receptor is involved in maintaining the autonomous growth of cultured hepatoma cells in vitro. Moreover, the block of this autocrine loop with the neutralizing antibody against HPO can partially inhibit the growth of hepatocytes. These results give suggestion to us that HPO may play an important role in the growth of hepatoma. Researches also indicated that the distribution of HPO in hepatoma cells is positively related with the expression of c-Met and the invasiveness of cells, that is to say, HPO is overexpressed in HepG2 and HLE hepatocellular cell lines with high level c-Met and high invasion ability, whereas HPO is lower expressed in SMMC-7721 and Bel-7402 hepatoma cell lines with low invasion ability.On the basis of the above researches, the relationship between HPO and the invasiveness of hepatoma was studied with Bel-7402 hepatoma cell line in which HPO is low expressed, and the roles of the two types of HPO (their molecular weight are 15kDa and 23kDa respectively) were studied at thesame time. HPO and HPO-205 genes were acquired from cDNA library of fatal liver with PCR methods and cloned into pMD18-T vector, and the sequences were identified. Then the eukaryotic expression vectors of HPO and HPO-205 were constructed by subcloning the genes into pcDNA3.1 (+) vector. The recombinant plasmids were transfencted into Bel-7402 hepatoma cell line, and the monoclonal cells high expressing HPO or HPO-205 stably were selected and identified. The effects of HPO and HPO-205 on the invasiveness and scattering ability of Bel-7402 cells were investigated and the expression of genes closely related with the regulation of hepatoma invasiveness was also researched, including c-Met, MMPs, u-PA and Ets-1.The results indicated that overexpression of HPO could up-regulate the expression of c-Met gene in Bel-7402 cells. However, only HPO itself could not influence the expression of MMP2, MMP9 and Ets-1 genes in Bel-7402 cells. Furthermore, overexpression of HPO could not result in the alteration of invasiveness and scattering ability of Bel-7402 cells. In addition, the expression of u-PA gene was up-regulated by HPO overexpression. These results suggest that HPO has close connections with the invasiveness of hepatoma. HPO might enhance the reactivity of hepatoma cells to HGF and increase their invasion ability by up-regulating the expression level of c-Met gene; and it might also promote the invasiveness of hepatoma cells by up-regulating the expression of u-PA. The results suggest that HPO may synergize with other cytokines such as HGF to promote the invasive growth of hepatoma cells. Further studies need to be done on this aspect.The results also indicated that HPO-205 has similar function as HPO on the invasion and scattering ability of Bel-7402 cells. This suggests that HPOand HPO-205 have same biological activities in hepatoma cells, and the variation of their N-terminus may be related to their location in cell compartments. Under some uncertain mechanisms, HPO-205 in cytosol is degraded into HPO, which is composed of carboxyl-terminus 125 amino acids of HPO-205 and translocates into nucleus.Conclusions:1. overexpression of HPO could up-regulate the expression of c-Met gene in Bel-7402 cells.2. Expression of u-PA gene was up-regulated by HPO overexpression in Bel-7402 cells.3. HPO itself could not influence the expression of MMP2, MMP9 and Ets-1 genes in Bel-7402 cells.4. HPO and HPO-205 have same biological activities in hepatoma cells.5. HPO is closely related with the invasion ability of hepatoma cells.