Effects Of AFP Gene Silencing On Migration And Adhesion Of Hepatocellular Carcinoma

Background and Objective: Primary liver cancer(PLC)is one of the most common alimentary canal malignancies in the world,which has a poor prognosis with an increase 600,000 cases every year.Most patients reach an advanced stage at diagnosis because of its insidious onset,which results in a high mortality.So it is urgent to find an effective method to treat PLC.AFP(alpha fetoprotein)is a kind of glycoprotein that originates from embryonic endodermal cells.It has a high expression in serum of most patients suffering from hepatocellular carcinoma,and in serum of a minority of patients suffering from gastric carcinoma,pancreatic carcinoma and reproductive system carcinoma.It is generally utilized as a tumor marker that is helpful for diagnosis of hepatocellular carcinoma(HCC)and prediction of HCC recurrence.At current,more and more researches have indicated that AFP plays an important role in proliferation,apoptosis and invasion,our research aimed to observe the effects of AFP gene silencing on migration and adhesion of hepatocellular carcinoma cell line EGHC-9901 and its mechanism.Methods: First we constructed the plasmid p Silencer3.0-H1-AFP according to the instructions of the literature,and then transfected it into the cell line EGHC-9901,then these cells were divided into three groups: the experimental group(transfected with AFP-si RNA),the vector control group(transfected with vector),and the blank control group(without treatment).Then transwell assay was used to test cell chemotactic migration ability;the cell aggregation assay was used to analyze alteration of homogeneous adhesion ability whereas Laminin adhesion assay was applied to evaluate its heterogeneous adhesion ability;Western blotting was applied to detect the expression of migration and adhesion-related proteins E-cadherin,CD44V6,CD15,integrin ?5,?-catenin,and ?-catenin.Results: 1.Transwell assay demonstrated more cells of the experimental group(132±16),that migrated through the polycarbonate membrane than those of the vector control group(335±16)and blank control group(371±32),P<0.05 through statistical analysis.2.Cell aggregation assay indicated that aggregation index(AI)of the experiment group was 0.263±0.010,P>0.05 through statistic analysis,versus that of the vector control group was 0.216±0.013 and the blank group was 0.220±0.009 when the incubation time was 20 min;but this assay witnessed markedly higher aggregation index of the experimental group(0.530±0.020 and 0.632±0.031),P<0.05 through statistic analysis,versus that of the vector control group(0.223±0.017 and 0.322±0.021)and the blank control group(0.240±0.012 and 0.376±0.019)when the incubation time was 40 min and 60 min.3.When we cultured cells for 30 min,the OD value of the experimental group was 0.225±0.014,P>0.05 through statistic analysis,versus that of the vector control group(0.223±0.018)and blank control group(0.267±0.020);When we cultured cells for 60 min,the OD value of the experimental group was 0.157±0.008,P<0.05 through statistic analysis,versus that of the vector control group(0.347±0.026)and blank control group(0.363±0.039),respectively,with statistically significant difference.At 90 min,the OD value of the experimental group was 0.139±0.010,P<0.05 through statistic analysis,versus that of the vector control group(0.476±0.023)and blank control group(0.503±0.045),respectively,with statistically significant difference.4.The expression levels of integrin ?1 protein in the experimental group,vector control group and blank control group were 0.337,0.078,and 0,051,respectively,P<0.05,the expermental group vs vector control group or blank group through statistic analysis;the expression levels of CD15 protein in the experimental group,vector control group and blank control group were 0.221,0.629,and 0.631,respectively,P<0.05,the experimental group vs vector control group or blank group through statistic analysis;But no significant statistical difference was found between the three groups relevant to the expression levels of E-cadherin protein(experimental group:0.883,vector control group:0.896,blank control group:0.873);CD44V6 protein(experimental group:0.627,vector control group:0.596,blank control group:0.678);Integrin ?5 protein(experimental group:0.433,vector control group:0.392,blank group were:0.403);?-cateinin protein(experimental group:0.368,vector control group:0.401,blank control group: 0.413);?-cateinin protein(experimental group: 0.563,vector control group:0.492,blank control group: 0.513),P>0.05 through statistic analysis.Conclusion: Silencing of AFP gene may inhibit chemotactic migration ability of hepatoma carcinoma cells,promote homogeneous adhesion between hepatoma carcinoma cells and inhibit heterogeneous adhesion to other tissues or organs partly by up-regulating the integrin ?1 and down-regulating CD15 expression.Our research on migration and adhesion of hepatocellular carcinoma cells after silencing of AFP gene will be helpful for further exploring the role of AFP in invasion and metastasis of HCC.