Constructing a Novel Hypoxia-Inducible Bidirectional shRNA Expression Vector for Simultaneous Gene Silencing in Colorectal Cancer Gene Therapy

Nonspecific siRNA expression limits its application in cancer gene therapy. Therefore, a tightly regulated and reversibly inducible RNAi system is required to conditionally control the gene expression. This investigation aims at constructing a hypoxia/colorectal tumor dual-specific bidirectional short hairpin RNA (shRNA) expression vector. First, carcinoma embryonic antigen (CEA) promoter designed in two directions. Then, pRNA-bipHRE-CEA vector was constructed by insertion of the vascular endothelial growth factor enhancer between two promoters for hypoxic cancer-specific gene expression. To confirm the therapeutic effect of the dual-specific vector, two shRNA oligonucleotides were inserted in the downstream of each promoter. QRT-polymerase chain reaction and western blot assays were performed to estimate the mRNA and protein expression levels. Both mRNA and protein levels were significantly reduced (50%-60%) in the hypoxic colorectal cancer-treated cells when compared with the controls. The novel bidirectional hypoxia-inducible shRNA expression vector may be efficient in colorectal cancer-specific gene therapy.