Abstract:

Objective To construct recombinant adenovirus vectors carrying Ad.RGD-P53 and/or Ad.RGD-ING4-P53 based on constructed empty vector Ad.RGD and adenovirus Ad.RGD-ING4 and explore the inhibition effect on human lung adenocarcinoma cells. Methods The P53 gene was obtained by PCR and pAd.RGD-P53 and pAd.RGD-ING4-P53 homologous recombinant adenovirus plasmids were constructed. Plasmids were packaged and amplified by using QBI-293A cell and the titer was detected. A549 cells were infected by Ad.RGD, Ad.RGD-ING4, Ad.RGD-P53, and Ad.RGD-ING4-P53, respectively. The efficiency of infection of recombinant adenoviruses towards A549 cells was detected by flow cytometry. Protein expressions of ING4 and/or P53 in A549 and PC-9 cells were detected by Western blotting. The growth of A549 cells was detected by MTT and the apoptosis of A549 and PC-9 cells were detected by flow cytometry. The mRNA expressions of apoptosis related genes Caspase-3, BAX, and BCL-2 in A549 cells were detected by real-time PCR. Results The results of PCR and enzyme digestion showed that pAd.RGD-P53 and pAd.RGD-ING4-P53 homologous recombinant adenovirus plasmids were successfully constructed. The titer detection reached 1×10⁹-1×10¹⁰ pfu/mL after being packaged and amplified. The efficiency of infection of recombinant adenoviruses towards A549 cells was 90%-95%. Results of Western blotting showed that ING4 and/or P53 protein successfully expressed in A549 and PC-9 cells. MTT found that ING4 and/or P53 recombinant adenoviruses significantly inhibited the growth of A549 cells. Flow cytometry confirmed that expressions of ING4 and/or P53 could induce the apoptosis of A549 and PC-9 cells. The induction of apoptosis by double gene recombinant adenoviruses was more significant than that of single gene recombinant adenoviruses (P<0.05). Results of real-time PCR showed that expressions of ING4 and/or P53 induced by adenoviruses up-regulated mRNA expressions of Caspase-3 and BAX in A549 cells and down-regulated mRNA expression of BCL-2. The regulation of expressions of Caspase-3, BAX, and BCL-2 by double gene recombinant adenoviruses was more significant than that of single gene recombinant adenoviruses (P<0.05). Conclusion The Ad.RGD-P53 and Ad.RGD-ING4-P53 recombinant adenoviruses were constructed successfully. Expressions of target genes of recombinant adenoviruses can obviously inhibit the growth of human lung adenocarcinoma cells and induce its apoptosis. The effects of double gene recombinant adenoviruses are more significant than those of single gene recombinant adenoviruses. The molecular mechanism may be relevant to up-regulation of expressions of apoptosis related genes Caspase-3 and BAX and down-regulation of expression of BCL-2.

Key words: RGD adenovirus, ING4 gene, P53 gene, human lung adenocarcinoma cell