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Cloning and expression of human vascular endothelial growth factor gene and inhibition of its expression by antisense
M Yazdanfar1, M Bandehpour, F Yarian
1Islamic Azad University, Research and Sciences Campus.
Background And The Purpose Of The Study:
Angiogenesis is an important process in physiology and disease pathogenesis and is controlled in a healthy body by a number of stimulatory and inhibitory factors. The aim of this study was to determine the effect of antisense transcript on the sense transcript of the endothelial growth factor (EGF) gene in bacterial system as an approach for the gene regulation in tumors.
Methods:
The hepatoma cell line (HepG2) was stimulated by PMA. VEGF mRNA was used for RT-PCR. VEGF cDNA was synthesised and cloned into T-vector pTZ57R, then sense fragment of VEGF subcloned into pACYC Duet-1 expression vector and antisense VEGF subcloned into pCDNA3 expression vector. Recombinant plasmids were transforemed into BL21 bacterial cells. Expression of recombinant plasmid was analysed by western blot technique.
Results:
The recombinant pCDNA3-VEGF (pYZantiVEGF) was successfully expressed in BL21 cells. Western blot analysis showed that the expression of VEGF decreased significantly in the cells transfected with VEGF antisense RNA compared with the pACYCDUET-1-VEGF (pYZsenseVEGF) transfected and control.
Major Conclusions:
The expression of VEGF in BL21 cells was strong. In vitro, antisense of VEGF inhibited VEGF expression significantly in BL21 cells.
Insights
Antisense RNA targeting vascular endothelial growth factor (VEGF) significantly inhibited its expression in bacterial cells. This study demonstrates a bacterial system for gene regulation, offering a novel approach for tumor research.
Area of Science:
- Molecular Biology
- Gene Regulation
- Biotechnology
Background:
- Angiogenesis is crucial in physiological and disease processes.
- Gene expression is tightly regulated by stimulatory and inhibitory factors.
- Tumor growth is often associated with aberrant angiogenesis.
Purpose of the Study:
- To investigate the effect of antisense RNA on vascular endothelial growth factor (VEGF) sense transcript.
- To establish a bacterial system for gene regulation as a potential anti-tumor strategy.
Main Methods:
- VEGF mRNA was isolated from HepG2 cells and converted to cDNA.
- Sense and antisense VEGF cDNA fragments were cloned into expression vectors.
- Recombinant plasmids were transformed into E. coli BL21 cells for expression analysis via Western blot.
Main Results:
- Successful expression of recombinant VEGF antisense RNA (pYZantiVEGF) in BL21 cells.
- Significant reduction in VEGF expression in cells treated with antisense RNA compared to sense RNA and control.
Conclusions:
- Bacterial expression of VEGF was confirmed.
- In vitro, VEGF antisense RNA effectively inhibited VEGF gene expression in BL21 cells.
