Hypoxia-inducible bidirectional shRNA expression vector delivery using PEI/chitosan-TBA copolymers for colorectal Cancer gene therapy
| Autor: | Bita Javan, Fatemeh Atyabi, Majid Shahbazi |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Polymers Genetic enhancement Genetic Vectors Apoptosis General Biochemistry Genetics and Molecular Biology Small hairpin RNA 03 medical and health sciences 0302 clinical medicine RNA interference Gene expression Gene silencing Humans Sulfhydryl Compounds General Pharmacology Toxicology and Pharmaceutics Particle Size RNA Small Interfering Hypoxia beta Catenin Chitosan Expression vector Chemistry Cell Cycle Gene Transfer Techniques General Medicine Transfection Genetic Therapy Genes bcl-2 030104 developmental biology 030220 oncology & carcinogenesis Cancer cell Cancer research Nanoparticles Colorectal Neoplasms HT29 Cells |
| Zdroj: | Life sciences. 202 |
| ISSN: | 1879-0631 |
| Popis: | Aims This investigation was conducted to construct a hypoxia/colorectal dual-specific bidirectional short hairpin RNA (shRNA) expression vector and to transfect it into the colon cancer cell line HT-29 with PEI/chitosan-TBA nanoparticles for the simultaneous knock down of β-catenin and Bcl-2 under hypoxia. Main methods To construct a pRNA-bipHRE-CEA vector, the carcinoma embryonic antigen (CEA) promoter designed in two directions and the vascular endothelial growth factor (VEGF) enhancer were inserted between two promoters for hypoxic cancer specific gene expression. To confirm the therapeutic effect of the dual-specific vector, β-catenin and Bcl-2 shRNAs were inserted downstream of each promoter. The physicochemical properties, the cytotoxicity, and the transfection efficiency of these PEI/chitosan-TBA nanoparticles were investigated. In addition, the antitumor effects of the designed vector on the expression of β-catenin and Bcl-2, cell cycle distribution, and apoptosis were investigated in vitro. Key findings The silencing effect of the hypoxia-response shRNA expression vector was relatively low (18%–25%) under normoxia, whereas it was significantly increased to approximately 50%–60% in the HT-29 cell line. Moreover, the cancer cells showed significant G0/G1 arrest and increased apoptosis due to gene silencing under hypoxia. Furthermore, MTS assay, fluorescence microscopy images, and flow cytometry analyses confirmed that the PEI/chitosan-TBA blend system provided effective transfection with low cytotoxicity. Significance This novel hypoxia-responsive shRNA expression vector may be useful for RNA interference (RNAi)-based cancer gene therapy in hypoxic colorectal tumors. Moreover, the PEI/chitosan-TBA copolymer might be a promising gene carrier for use in gene transfer in vivo. |
| Databáze: | OpenAIRE |
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