Effects And Mechanism Of Silencing AK4 On Proliferation,Migration And EMT Of Intrahepatic Bile Duct Carcinoma Cell

Objective(s): To investigate the effects of silencing adenylate kinase 4 on the proliferation,migration and EMT of intrahepatic bile duct cells and the changes of molecular mechanism.Methods:Immunohistochemistry was used to determine the difference of AK4 expression between normal liver tissue and intrahepatic cholangiocarcinoma.Three strains of intrahepatic bile duct cancer cells(HCC-9810,RBE,HUCCT1)were selected.AK4 high-expression cell lines were screened out by Real-time quantitative PCR and western blotting.AK4 expression in cells was detected by immunofluorescence technique.The expression of AK4 in cell lines was silenced by small interfering RNA technology.The transfection efficiency was verified by fluorescence microscopy and WB technology,and the si RNA with the best silencing effect was selected.The changes of cancer cell migration ability after si RNA silencing AK4 were detected by scratch assay.Ed U assay was used to detect the changes of cancer cell proliferation after si RNA silencing AK4.The changes of EMT-related proteins(E-cadherin,Fibronectin,Snail 2and Vimentin)after AK4 silencing were detected by WB.Wnt/β-Catenin pathway proteins(β-Catenin,Cyclin D1,MMP-2,c-Myc and Survivin)were detected by WB after AK4 silencing.The experimental data were statistically analyzed using IBM SPSS Statistics 26,STATA 14 and Graph Pad Prism 8.0.Results: Immunohistochemistry(IHC)revealed AK4 significantly higher expression in Intrahepatic Cholangiocarcinoma(ICC)tissues.RT-q PCR and WB results showed that HUCCT1 cells had the highest AK4 expression among HCC-9810,RBE and HUCCT1 cells,so HUCCT1 were used for follow-up experiments.Immunofluorescence assay showed that AK4 was mainly distributed in the cytoplasm.Fluorescence microscopy and WB showed high transfection efficiency of si RNA,and WB showed the best silencing effect of si RNA-AK4-3,so it was used in the subsequent steps of this experiment.Scratch test showed that HUCCT1 migration ability was significantly weakened in si RNA-AK4-3 group(P < 0.05).Ed U results showed that HUCCT1 proliferation ability was significantly decreased in si RNA-AK4-3 group(P< 0.05).EMT experiment showed that in the si RNA-AK4-3 group,the expression level of epithelioid markers E-cadherin in HUCCT1 cells was significantly increased,while the interstitial markers Fibronectin,Snail 2 and Vimentin were significantly decreased(P < 0.05).WB detection of Wnt/β-Catenin channel shows: The key protein of signaling pathway β-Catenin was significantly silenced in si RNA-AK4-3 group,and the downstream proteins Cyclin D1,MMP-2 and c-Myc also showed decreased expression after silencing AK4,with statistical significance(P < 0.05).There was no difference in Survivin protein between NC group and experimental group(P > 0.05).Conclusion(s): The high expression of AK4 is one of the factors that promote the proliferation,metastasis and EMT of intrahepatic bile duct cell carcinoma.AK4 promote the proliferation,migration,EMT and other biological behaviors of HUCCT1 cells may through the Wnt/β-Catenin signaling pathway.