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| The Inhibition of Ad. RGD-ING4-PTEN on MEG01 Human Leukemia Cell |
| HUANG Chen, WANG Jia-rong, YANG Ji-cheng, SHENG Wei-hua, MIAO Jing-cheng |
| Medical College of Soochow University, Cell and Molecular Biology Institute, Suzhou 215123, China |
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Abstract Objective: To construct a recombinant adenoviral vector modified by RGD-4C co-expressing inhibitor of growth 4 ( ING4 ) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) and to study its inhibitory effects on human leukemia cell line MEG01. Methods:The recombinant adenovirus Ad.RGD-ING4-PTEN was obtained by gene recombination and in vitro packaging technique. Firstly, between the restriction enzyme cutting sites NotⅠand XhoⅠ, the PTEN fragment were inserted respectively, at the foundation of pAdTrack-CMV-ING4-polyA-promoter and pAdTrack-CMV-PolyA-promoter. Secondly, the transfered vector linearized by Pme I digestion and the backbone vector modified by RGD-4C were further co-transformed into the bacteria BJ5183 for homologous recombination. Lastly, the QBI-293A cell was used for packaging and amplification. The human leukemia cell line MEG01 was infected with Ad.RGD-ING4-PTEN. The infection efficiency of adenovirus to MEG01 was detected by flow cytometry. The expression of ING4 and PTEN was detected by Western blot. Cell growth inhibition was detected by CCK-8 assay. The cell apoptosis and the cell cycle were detected by flow cytometry. Results: The results of identification test show that the successful build of Ad.RGD-ING4-PTEN,the adenovirus which can express the double gene of ING4 and PTEN.It can infect MEG01 cell line effectively. The expression of ING4 and PTEN in MEG01 is obvious; ING4 and PTEN gene can significantly inhibit the growth of MEG01, induce its apoptosis and produce cycle arrest, and the fuction of double gene combination is more obvious than Single gene. Conclusion:Adenoviral vector modified by RGD-4C co-expressing ING4 and PTEN were successfully constructed. RGD-4C can significantly improve the efficiency of the infection to human leukemia cell line MEG01. Ad.RGD-ING4-PTEN can inhibit the cell growth of MEG01 and induce its apoptosis and block the cell cycle.
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Received: 24 December 2013
Published: 25 March 2014
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