Conditionally replicating adenovirus activated by CXCR4 promoter in lung cancer |
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| Authors: | LI Long-guang LI Shu-hua WANG Hong-yan LONG Jie XIE Xiao-bin ZHANG Ya-jie |
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| Institution: | Department of Pathology, Guangzhou Medical University, Guangzhou 510182, China |
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| Abstract: | AIM: To construct a conditionally replicating adenovirus vector activated by CXCR4 promoter and to evaluate its ability of lysing the lung cancer cells specifically. METHODS: Human CXCR4-E1A gene amplified by PCR was cloned into the shuttle plasmid pDC316-GFP to construct the recombinant shuttle plasmid pDC316-CXCR4-GFP. The recombinat shuttle plasmid and adenovirus genomic plasmid pBHG-lox-E1, 3Cre were transfected into 293 cells to construct the recombinant adenovirus CRAd-CXCR4-GFP. PCR was used to detect the target gene fragments, and the viral titer was determined. A549 cells with the highest mRNA expression of CXCR4 were screened out from 5 kinds of lung cancer cell lines by real-time PCR. CXCR4 promoter activity and adenovirus replication numbers were detected in A549 cells after transfection of CRAd-CXCR4-GFP and Ad-NULL. CRAd-CXCR4-GFP and Ad-NULL were transfected into A549 cells and 16HBE cells, the apoptotic rates were detected by flow cytometry and the viability was analyzed by CCK-8 assay. RESULTS: The recombinant plasmid pDC316-CXCR4-GFP was constructed successfully. Green fluorescence was observed in 293 cells under fluorescent microscope after co-transfection of pDC316-CXCR4-GFP and pBHG-lox-E1, 3Cre at 11 d. Green fluorescence was observed in 293 cells after infection of amplified 3rd generational adenovirus. PCR showed that the purpose gene was successfully integrated in recombinant adenovirus genome. The virus in the supernatant reached a titer of 1×1013 PFU/L. The mRNA expression of E1A and E4 in the A549 cells after transfection of CRAd-CXCR4-GFP was markedly increased compared with Ad-NULL group. Compared with Ad-NULL group and empty control group, the apoptotic rate and the viability of A549 cells in CRAd-CXCR4-GFP group had no significant difference in the first 4 d, the apoptotic rate increased significantly at 5 d, and the cell viability declined significantly at 5 d, but the apoptotic rate and the viability of 16HBE cells in each group had no significant difference within 5 days. CONCLUSION: The conditionally replicating adenovirus vector CRAd-CXCR4-GFP has been successfully constructed, which has the ability of lysing lung cancer cells specifically. |
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| Keywords: | Conditionally replicating adenovirus Tumor-specific promoter CXCR4 promoter |
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