Abstract: Objective To study the effect of pluripotency factor Nanog on the expression of the cell cycle related proteins,and then to explore its effect on the proliferative ability of HepG2 cells.Methods TALENs gene editing tool was employed to induce mutation and downregulation expression of Nanog.T7 endonuclease 1 and genomic sequencing was used to analyze the mutation efficiency of Nanog.RT-PCR and western blot were used to determine the expression of mRNA and protein of Nanog,respectively.Real-time cell based assay system was used to measure the proliferative ability of wild-type HepG2 cells and monoclonal HepG2 cells with Nanog mutation.Results TALENs successfully induced mutation of Nanog gene.The targeting efficiency of mixed cells was analyzed by T7 endonuclease 1 approached 40% after two transfection with plasmid of Nanog-TALENs.Additionally,the Nanog mRNA expression level of monoclonal HepG2 with Nanog mutation was downregulated by 3.4 times compared to the wild type HepG2 cells,and the Nanog protein expression level was downregulated by 3.6 times.The cell cycle related proteins CyclinD1/D3,CyclinE1 and CDK2 expression were downregulated in monoclonal HepG2 with Nanog mutation in comparison to the wild type HepG2 cells.Conclusion Nanog plays a role in influencing the proliferative ability of HepG2 cells through modulating the expression of the cell cycle related proteins CyclinD1/D3,CyclinE1 and CDK2.The downregulation expression of Nanog can inhibit the proliferative capacity of HepG2 cells via the regulation of the cell cycle related proteins.

Key words: TALENs, Nanog, HepG2 cells, Hepatocellular carcinoma