| Objectives Augmenter of liver regeneration (ALR), a heat-stable hepatotrophic growth factor, was first discovered and cloned from rat livers. Its action is different from hepatocyte growth factor (HGF). In Preliminary study we found that ALR could stimulate hepatoma proliferation directly by autocrine, the appropriate dose of ALR can be used as an immune inhibitor of the immune system to evade the immune response of hepatocellular carcinoma. When the level of expression hALR of nucleic acid and protein were inhibited, the liver tumor were both inhibited significantly in vivo and vitro.There are complicated cellular network , multi-gene involvement in the occurrence and development of tumor . Transforming growth factor-α(TGF-α) takes part in cell growth and transformation, combined with the epidermal growth factor receptor (EGFR) cause cell proliferation and differentiation. Many malignant tumors were both of the high TGF-αexpression and EGFR expression, tumor cell autocrine TGF-αreceptor on their membranes to form a loop of its own proliferation of tumors plays an important role in the occurrence and development. The number and affinity of ALR receptor in hepatocytes were lower than in hepatomaes. ALR can up-regulate the expression of EGFR mRNA, through the role of the EGFR to regulate regeneration of liver cells.EGFR can mediate urokinase-type plasminogen activator receptor (uPAR) / integrin / fibronectin-induced growth, thereby promoting tumor cell growth and metastasis, EGFR kinase (to hydrolysis EGFR) can block uPAR-induced growth of channels. UPAR is a cell surface anchor protein receptor, can affect the invasion and metastasis of tumor cells after stimulating. This suggests that the interaction between cytokines may play a role in the tumor development process.This study was to investigate the effect of blocking the expression of hALR on the expression of TGF-αand EGFR of HCC cell line HepG2 with small interfering RNA (siRNA) targeting ALR. This study furthered an understanding of the mechanism of carcinogenesis of hepatocellular carcinoma.Methods⒈The expressing siRNA plasmid pSIALR—A targeting hALR and the unrelated control plasmid pSIALR-B were constructed successfully and transfected into HepG2 cells with lipofectamine 2000 methods ,respectively.the expression of green fluorescent protein was observed under a fluorescent microscope to calculate transfection efficiency. ⒉The protein level of hALR in the transfected cells was measured by immunocytochemistry to determine the inhibitory effect.⒊According to different plasmid transfection, HepG2 cells were divided into three groups: transfection group (transfected pSIALR-A), the control group (transfected pSIALR-B), blank control group (non-transfected with the recombinant plasmid). there are three duplicated wells in each group. The expression of TGF-αin the cell culture supernatant was detected by radioimmunoassay. The expression of EGFR was detected by Western blot. Each experiment was repeated for three times.Resluts1. At 24 hours after transfection, there are a large number of bright green fluorescent protein (GFP) expression in HepG2 cells .There are are more than 80% of the cells which has the expression of green fluorescent protein after transfected by the two plasmids.The transfection efficiency is high. The two groups of cells have basically the same transfection efficiency.2. At 48 hours after transfection, the protein level of hALR was measured with immunocytochemistry. In HepG2 cells which transfected pSIALR-A , the level of hALR protein expression decreased significantly. HepG2 cells which transfected the negative control plasmid pSIALR-B still has the expression of hALR. In the level of protein ,it suggests that siRNA targeting hALR can significantly inhibit the expression of hALR.3. The expression level of TGF-αin the transfection group is significantly lower than the blank group and the control group (P <0.05).4. The relative quantity of EGFR expression of the transfection group,the blank group, the control group detected by Western blot was 1.115±0.606,1.131±0.509,0.946±0.136,respectively.The relative quantity of EGFR in the transfection group is significantly lower than the blank group and the control group (P <0.05).Conclusion1. The expressing siRNA plasmid pSIALR—A targeting hALR and the unrelated control plasmid pSIALR-B were transfected into HepG2 cells with lipofectamine 2000 methods,respectively.The transfection efficiency is high.2. The pSIALR-A inhibited the expression of hALR in HepG2 cells significantly as compared with pSIALR-B.3. The Expression of Transforming Growth Factor-α(TGF-α) and Epidermal Growth Factor Receptor in HepG2 was inhibited after transfection in vitro. |
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