Relationship Between Augmenter Of Liver Regeneration And Carcinogenesis Of Hepatocellular Carcinoma

Objective: Augmenter of liver regeneration (ALR), a heat-stable hepatotrophic growth factor, was first discovered and cloned from neonatal rat livers. Although its action is similar to hepatocyte growth factor (HGF) in promoting liver regeneration, its molecular structure is different from HGF. Present research indicated ALR could stimulate the proliferation of hepatoma cells in a dose-dependent manner in vitro. Moreover, ALR could increase the proliferation of cells and protect cells against damage after ALR expression plasmid was transfected into hepatoma cells. It was also showed by immunohistochemistry that the staining of ALR was strongly positive in the liver tissue of patients with hepatocellular carcinoma. They all demonstrated that ALR was related to the carcinogenesis of hepatocellular carcinoma. Moreover, we also observed ALR could inhibit the proliferation of mononuclear cells induced by ConA or specific antigens in vitro. Why hALR was highly expressed in hepatocellular carcinoma? We suspected ALR might play an important role in the carcinogenesis of hepatocellular carcinoma because of being involved in autocrine growth of hepatocellular carcinoma and immune suppression in hepatocellular carcinoma. However, the association of ALR and carcinogenesis of hepatocellular carcinoma was not fully understood. Our objective in this study was to investigate that biological behavior of hepatocellular carcinoma was influenced by siRNA targeting hALR and anti-ALR McAb on nuclei acid and protein level in vitro and in vivo. In this study, the RNAi plasmid pSIALR-A and the unrelated control plasmid pSIALR-B were transfected into HepG2 cells respectively. After transfection, the expression of hALR was measured. The proliferation of HepG2 cells after treated with pSIALR-A and anti-hALR McAb was detected. The growth of the xenograft tumor was observed after being treated with pSIALR-A and anti-hALR McAb in nude mice. The effect of rALR on proliferation of rat peripheral blood mononuclear cells was detected after rat peripheral blood mononuclear cells were activated by membrane of HepG2 cells. This study not only furthered an understanding of the mechanism of carcinogenesis of hepatocellular carcinoma but also provided potential candidates for gene therapy of hepatocellular carcinoma. Methods: 1. Expression of hALR in hepG2 cells: The expression of hALR in HepG2 cells was observed with immunocytochemistry on protein level. The expression of hALR in HepG2 cells was detected with RT-PCR on nuclei acid level. 2. To observe effects of anti-hALR McAb on biological behavior of hepatocellular carcinoma: 1) The proliferation of HepG2 cells treated with anti-hALR McAb was detected by 3H-TdR incorporation approach. 2) HepG2 cells were resuspended in 1:500 anti-hALR McAb and 1:500 ascites induced by SP2/0 cells, respectively (anti-hALR McAb/HepG2 cells and ascites induced by SP2/0 cells /HepG2 cells). Subcutaneous tumors in nude mice were induced by inoculation anti-hALR McAb/HepG2 cells and ascites induced by SP2/0 cells /HepG2 cells. Every other day, each group of mice were treated with 1:500 anti-hALR McAb or 1:500 ascites induced by SP2/0 cells by i.p. The growth of the xenograft tumor was observed after inoculation. The expression of hALR in thexenograft tumor was observed with immunocytochemistry. 3. Construction and identification of expressing siRNA plasmid of hALR and unrelated control plasmid pSIALR-B: After being constructed successfully, the expressing siRNA plasmid pSIALR-A, which targeted the cDNA of hALR, and the unrelated control plasmid pSIALR-B were transfected into HepG2 cells respectively (After transfection, the cells were nominated pSIALR-A / HepG2 and pSIALR-B / HepG2). At 24 hours after transfection, the cells were examined for the presence of GFP by an inverted fluorescent microscope. At 48 hours after transfection, the protein level of hALR was measured with immunocytochemistry; Meanwhile, the reverse transcriptive PCR (RT-PCR) was performed to detect the expression of hALR mRNA. 4. To observe...