The effect of early B cell factor 3 on HepG2 cell cycling / 中国免疫学杂志

ABSTRACT

Objective:To clone the encoding sequence of human EBF3 gene,construct recombinant eukaryotic expression plasmid vector pEGFP/EBF3,and study the effect of EBF3 on HepG2 cell cycling.Methods:Total RNA was isolated from placental tissue.Full-length human EBF3 cDNA was amplified by RT-PCR,cloned into eukaryotic expression plasmid vector pEGFP-N1 and sequenced.The expression and sub-cellular localization of the fusion protein EBF3-EGFP in HepG2 cells were analyzed by Western blot.Cell cycles were analyzed with flow cytometry analysis.Results:Obtained full encoding sequence of early B cell factor 3 was identical with that included in GeneBank,and the eukaryotic expression plasmid vector pEGFP/EBF3 was constructed correctly.24 h after transfected by pEGFP/EBF3,the fusion protein EBF3-EGFP was observed mainly in the cellular nucleus under the inverted fluorescence microscope.Western blot analysis confirmed that the EBF3-EGFP fusion proteins of Mr 87 000 were detected in both cytoplasmic and nuclear protein of the HepG2 transfected by pEGFP/EBF3 for 24 h or 48 h.Flow cytometry analysis revealed that the percentage of cells in the S phase was markedly increased in HepG2 cells transfected by pEGFP/EBF3 as compared with that in pEGFP-N1 transfected cells.These findings suggested that transfection of EBF3 gene into HepG2 induced cell proliferation by increasing the number of cells from G1 phase to G2 phase.Conclusion:The recombinant eukaryotic expression plasmid vector pEGFP/EBF3 is successfully established.The percentage of cells in the S phase is markedly increased in pEGFP/EBF3 transfected cells as opposed to pEGFP-N1 transfected cells.It is likely that EBF3 promotes HepG2 cells proliferation through DNA replication.