1 Department of Molecular Medicine, School of Advanced Technologies in Medicine, Golestan University of Medical Sciences , Gorgan, Iran .
2 Medical Cellular & Molecular Research Center, Golestan University of Medical Sciences , Gorgan, Iran .
Cancer Biother Radiopharm. 2018 Apr;33(3):118-123. doi: 10.1089/cbr.2017.2401.
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.
非特异性 siRNA 表达限制了其在癌症基因治疗中的应用。因此，需要一种严格调控和可逆转诱导的 RNAi 系统来条件控制基因表达。本研究旨在构建一种缺氧/结直肠癌双特异性双向短发夹 RNA（shRNA）表达载体。
首先，设计了两个方向的癌胚抗原（CEA）启动子。然后，通过在两个启动子之间插入血管内皮生长因子增强子，构建了 pRNA-bipHRE-CEA 载体，用于缺氧条件下的肿瘤特异性基因表达。为了确认双特异性载体的治疗效果，在每个启动子的下游插入了两个 shRNA 寡核苷酸。通过 QRT-PCR 和 Western blot 检测来评估 mRNA 和蛋白表达水平。
与对照组相比，缺氧处理的结直肠癌细胞中的 mRNA 和蛋白水平均显著降低（50%-60%）。
新型双向缺氧诱导性 shRNA 表达载体可能在结直肠癌特异性基因治疗中有效。
