Response of hypoxia-response element in human liver cancer cells to different microenvironments
Objective: Bel-7402 cells were stably transfected with a vector constructed with multiple copies of the hypoxia response- element (HRE) sequence of the human vascular endothelial growth factor (VEGF) gene and with the enhanced green fluorescent protein (EGFP) to establish a human hepatoma cell line Bel-7402/5HRE-EGFP. This paper aimed to study the responses of HRE in human liver cancer cells to different microenvironments by observing the changes in hypoxia-inducible factor 1 (HIF-1α) and vascular endothelial growth factor (VEGF) expression in Bel-7402/5HRE-EGFP cell lines under hypoxic conditions. Methods: The expression vector was constructed with 5 copies of HRE sequence and a minimal cytomegalovirus (CMV) as promoter and green fluorescent protein (GFP) as a reporter gene. The effect of different microenvironments such as hypoxia, H2 O2 or acidic pH on the activity of HRE in Bel-7402 cells and the changes in the expression levels of HIF-1α and VEGF under hypoxic condition were determined by using flow cytometry and immunohistochemical staining method. The association of the staining intensity and the distribution of pimonidazole, a hypoxic probe, with the expression and the distribution of HIF-1α, VEGF, and GFP were analyzed. The influence of hypoxia in tumor tissues of nude mice on the activity of HRE and expression of related genes were observed. Results: The HRE in liver cancer cells was very sensitive to hypoxia, which induces up-regulation of HIF-1α and VEGF expressions in tumor cells or in tumor tissues. Both distribution regions of HIF-1α and VEGF were almost the same. Conclusion: Hypoxia plays a pivotal role in controlling the expression of angiogenesis-related factors in human liver cancer cells.