Targeting validation of a hypoxia-induced tumor-specific gene therapy vector

Objective: To verify the specificity and the response to hypoxia stimulation of a gene therapy vector containing the tumor suppressor gene CDX2 regulated by the hypoxia-induced enhancer (HRE) and the hTERT promoter.

Methods: (1) The tumor cell line LoVo was exposed to CoCl₂ (100, 200, 300, 400, and 500 μmol/L) for different time periods (1, 3, 5, and 7 d); cell viability and proliferation were detected by MTT method. (2) The recombinant lentiviral vector pLVX-5HRE-hTERTp-CDX2-3FLAG(5HhC) containing an hTERT promoter and 5 copies of the hypoxia-response elements (HRE) enhancer, which had been constructed in our previous study, was collected. 5HhC was then transfected into hTERT⁺ LoVo cells and hTERT^- HK-2 cells; the hTERT-specificity was verified by immunohistochemistry. The LoVo cells with stable expression of 5HhC (5HhC/LoVo) were obtained. Hypoxia microenvironment was simulated by CoCl₂, and Western blot and RT-PCR were employed to examine the expression of CDX2 regulated by hypoxia.

Results: As hypoxia prolonged, the viability and proliferation of the hTERT⁺ LoVo cells were inhibited by CoCl₂ of all concentrations, especially the higher concentrations (300, 400, and 500 μmol/L) (P<0.05). The hTERT⁺ LoVo cells were infected with the recombinant lentiviral vector 5HhC while the hTERT^- HK-2 cells were not infected. Western blot and RT-PCR confirmed that the expressions of CDX2 protein and mRNA in 5HhC/LoVo cells were further increased with hypoxia, especially with CoCl₂ of 300 μmol/L for 24 h.

Conclusion: Hypoxia microenvironment can upregulate the expression of CDX2 in the hTERT-specific gene therapy vector 5HhC regulated by the hypoxia-induced enhancer (HRE) and the hTERT promoter in hTERT⁺ LoVo cells.