1. Medical Cellular and Molecular Research Center, Golestan University of Medical Sciences, Gorgan 2. Medical Cellular & Molecular Research Center, Golestan University of Medical Sciences, Gorgan, Iran
Abstract
Introduction
Background: rna interference (rnai) is an efficient genetic approach in gene-specific knockdown and gene therapy; however, the imprecise expression of sirna limits its application in cancer gene therapy. therefore, a tightly regulated and reversibly inducible rnai system is required to conditionally control the gene expression in a temporal or spatial manner. this investigation aims at constructing a hypoxia/colorectal dual-specific bidirectional shrna expression vector.
Methods
Methods: a carcinoma embryonic antigen (cea) promoter with enhancer regulatory region was designed in two directions and vascular endothelial growth factor (vegf) enhancer was placed between the two promoters for hypoxic cancer specific gene expression. the 2700 nucleotide fragment was synthesized and subsequently cloned in prnat-u6.1/neo expression vector to construct prna-biphre-cea vector. the substitution of the u6 promoter into the bidirection cea core promoter was evaluated with double digestion analysis. to confirm the therapeutic effect of the dual-specific vector, two shrna oligonucleotides were inserted in the downstream of each promoter. qrt-pcr and western blot assays were performed to estimate the mrna and protein expression levels.
Results
Results: the construction of tumor-specific expression vector with two shrna was verified by double digestion test. both mrna and protein levels were significantly reduced (50-60%) in the hypoxic colorectal cancer treated cells when compared to the controls.
Conclusion
conclusion: our results suggested that the combination of the vegf gene's hre and the cea promoter specifically increased gene expression under hypoxia in colorectal cancer cell lines. therefore, the novel hypoxia-inducible sirna expression system has a potential tool for rnai based cancer gene therapy.
Keywords
Cea promoter, bidirectional promoter, shrna, tumor hypoxia, colorectal cancer
