Construction of a Plasmid Expressing MDA-7 Gene Modified by an iNGR Sequence and Its Evaluation for Apoptosis Induction in a Hepatocellular Cancer Cell Line

Authors
E. Zare 1
S. E. Hosseini 2
G. Mosayebi 1
M. Sadeghizadeh 3
M. Sadeghizadeh 3
T. Hashempour 4
S. Y. Hosseini 5
B. Khansarinejad 6
1 Department of Biotechnology, School of Medicine, Arak University of Medical Sciences, Arak, Iran
2 Gastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz , Iran
3 Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
4 Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
5 Gastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
6 Department of Microbiology and Immunology, Arak University of Medical Sciences, Arak, Iran
Abstract
Objective: Melanoma differentiation association gene-7 (MDA-7)/IL24 is a tumor suppressor gene. The iNGR peptide sequence, with excellent tropism to surface integrins has been employed for targeting of therapeutic molecules toward tumor cells. The purpose of our study was to construct a plasmid expressing modified MDA-7 fused with an iNGR peptide for better targeting to tumor cells.
Methods: At first, we amplified the MDA-7 sequence by PCR, while the reverse primer contained the iNGR peptide sequence to add it to the end of a new MDA-7 gene. The resultant MDA7-iNGR and MDA-7 were cloned into a pCDNA3.1 eukaryotic expression vector. The accuracy of cloning methods, integrity of the plasmids, and sequence were sequentially evaluated by digestions, colony-PCR, and sequencing. The expressions of the plasmid constructs were assayed by ELISA following their transfection into Ad-293 cells. Next, the plasmids were transfected into Hep-G2 cells and their mRNA were converted to cDNA. We assessed the gene expression levels of Gadd153 and Bax. As the final step, apoptosis induction of Hep-G2 cells following transfection was evaluated by the help of PI/Annexin V staining according to flow cytometry.
Results: The results showed the integrity of construct backbone in addition to reading frame of the MDA-7-iNGR sequence. A suitable expression/secretion of modified the MDA-7.iNGR protein was detected by ELISA assay of the culture supernatant when compared to the control construct that expressed unmodified MDA-7. The viabilty test demonstrated no benefit for this kind of modification of the MDA-7 protein. Real-time PCR and flow cytometry analyses revealed that the addition of iNGR to MDA-7 caused a decrease in its apoptotic effect on hepatic tumor cells compared to the normal protein. The modified protein had significant apoptosis induction compared to the negative control group (P<0.01).
Conclusion:Although the new pCDNA/MDA-7.iNGR plasmid expressed iNGR-fused MDA-7 protein efficiently, it could not improve the natural apoptosis property of normal MDA-7.

Keywords

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Pathobiology Reserach
Volume 19, Issue 4
September 2017
Pages 27-39