Aim: To construct the eukaryotic expression vector harboring transcriptional factor JunB and to observe the expression, localization and biological function of JunB in hepatic cancer cells.
Methods: The JunB gene was amplified by PCR from the human liver tissue cDNA library. After confirmed by DNA sequencing in the T-vector, the JunB gene was subcloned into pcDNA3.1(-) and pEGFP-C3 vectors, respectively. The recombinant vectors pcDNA-JunB and pEGFP-JunB were confirmed by Kpn I and BamH I restriction enzyme digestion. The recombinant vectors were transiently transfected into the hepatic cancer cells HepG2 using Lipofectamin2000. Western blotting was used to detect the expression of exogenous JunB and fluorescence microscopy was applied to observe the localization of JunB protein coupled with enhanced green fluorescent protein (EGFP) in hepatic cancer cells. Double luciferase reporter assay was performed to determine the effect of JunB on the transcriptional regulation of target genes.
Results: The recombinant eukaryotic expression vector carrying JunB or JunB-EGFP fusion gene was constructed and transiently transfected into HepG2 cells. Western blot analysis confirmed the exogenous expression of JunB. JunB-EGFP was observed uniquely in the nuclei. Double luciferase assay showed the transcriptional up-regulation of the VEGF in the presence of JunB.
Conclusion: We constructed the recombinant eukaryotic expression vectors harboring JunB and JunB-EGFP successfully. The exogenous JunB is localized in the nuclei of transiently transfected HepG2 cells, suggesting JunB may function as a transcriptional factor. The result of reporter assay showed that JunB up-regulates the expression of VEGF, a tumor-angiogenesis associated molecule, at a transcriptional level. These demonstrate that JunB might be a novel target for an anti-angiogenesis treatment for hepatic cancers.