AFP Induces Malignant Behaviors Of Liver Cells By Wnt/beta-catenin Pathway

Objective: HCC is one of the disease impacting on the health and life of human beings.In the occurrence and development of hepatocellular carcinoma,AFP is not only as a tumor marker,but also as a key factor which plays an important role in promoting the development of HCC.Many cellular signaling pathways plays a crucial role in the pathogenesis of HCC.And Wnt/beta-catenin signaling pathway paid much attention,which can promote the occurrence and development of malignant tumor,including lung cancer,colorectal cancer,liver cancer,gastric cancer and thyroid cancer in its abnormal state.However,it is not clear that the effect of abnormal AFP on normal liver cells.Therefore,this study discussed the effects on AFP overexpression in the malignant behavior of normal liver cell L02 and the relationship between AFP and Wnt/beta-catenin signaling pathway.Moreover it provided a new way for the clinical treatment that the further understanding of the pathogenesis of hepatocellular carcinoma.Methods: Normal human liver cells L02 was infected by lentivirus to establish L02-GFP and L02-AFP cells.After different concentrations of 5FU treatment of L02 cells in 24 hours,MTT assay was used to examine the survival rate of L02 cells to determine the appropriate concentration;L02,L02-GFP,L02-AFP cells were treated with corresponding concentration of 5FU in 24 hours and the inhibition rate of cell growth was calculated according to the absorbance.The cell scratch wound healing assay was used to study the scratch repair ability of L02,L02-GFP and L02-AFP cells.Transwell migration assay was used to detect the migration ability of L02,L02-GFP,L02-AFP cells.Western blot was used to analysis the expression of AFP,Src,beta-catenin and Cyclin D1 in L02,L02-GFP,L02-AFP and AFP-si RNA interference expression vector transfected L02-AFP cells.Results: AFP lentivirus expressed vector was successfully infected into L02 cells and successfully established a stable L02-AFP expression cell line(named L02-AFP).MTT results indicated that L02-AFP cells growth ratio significantly higher than L02 cells while treated with 5Fu.Transwell analysis displayed that the invasive numbers of L02-AFP cells were significantly higher than L02 cells.Scratch test results showed that the healing ratio of L02-AFP cells(52.15%±2.21)was significantly higher than that of L02 and L02-GFP cells(37.69%±2.21,40.39%±1.41).Western blotting results showed that AFP could promote the expression of Src,while interference the expression of AFP in L02-AFP cells,the expression of Src was inhibited.The expression of beta-Catenin and Cyclin D1 in L02-AFP cells lesser than L02 and L02-GFP cells.However,the expression of beta-Catenin and Cyclin D1 in L02-AFP cells were significantly suppressed while the cells were transfected with AFP-si RNA vectors.Conclusions: Established a stably AFP-expressed cell line,L02-AFP,which provided an experimental basis for the study of the effect of AFP on the malignant behaviors of L02 cells.The results showed that AFP was able to promote the proliferation,migration and drug-resistanance of normal liver cells,the role mechanism of AFP maybe involve in stimulating expression of Src,cyclin D1 via activating the Wnt/ beta-catenin signaling pathway.