Construction and identification of recombinant adenovirus with COX-2-shRNA

Objective To construct and identify the recombinant adenovirus with a short hairpin RNA(shRNA)targeting cyclooxygenase-2(COX-2),with a tag of enhanced green fluorescent protein(EGFP),and observe its effect on the proliferation of liver cancer SMMC-7721 cells.Methods The U6 expression promoter and shRNA sequences of previously constructed eukaryotic expression vector pGenesil-1-COX-2-shRNA and negative control plasmid pGenesil-1HK were subcloned into adenovirus shuttle plasmid pAdTrack.The constructed recombinant adenovirus shuttle plasmids pAdTrack-U6-COX-2-shRNA-EGFP and pAdTrack-U6-HK-EGFP were identified by enzyme digestion and DNA sequencing,then linearized with Pme Ⅰ and transformed to competent AdEasier.The obtained recombinant adenovirus plasmids pAd-U6-COX-2-shRNA-EGFP and pAd-U6-HK-EGFP were linearized with PacⅠ and transfected to AD293 cells for packaging.The obtained recombinant adenoviruses Ad-U6-COX-2-shRNA-EGFP and Ad-U6-HK-EGFP were subjected to three cycles of amplification and determined for titer.The mRNA transcription and protein expression of COX-2 in infected cells were determined by RT-PCR and Western blot.The effect of Ad-U6-COX-2-shRNA-EGFP on proliferation of SMMC-7721 cells was observed by MTS method.Results Recombinant adenovirus shuttle plasmids pAdTrack-U6-COX-2-shRNA-EGFP and pAdTrack-U6-HK-EGFP,as well as recombinant adenovirus plasmids pAd-U6-COX-2-shRNAEGFP and pAd-U6-HK-EGFP,were constructed correctly as proved by enzyme digestion and sequencing,and successfully transfected to AD293 cells.The titers of recombinant adenoviruses Ad-U6-COX-2-shRNA-EGFP and Ad-U6-HK-EGFP were 1.4 × 10 12 and 2.0 × 10 12 pfu / ml respectively.The mRNA transcription and protein expression of COX-2 as well as proliferation ability of SMMC-7721 cells infected with Ad-U6-COX-2-shRNA-EGFP were significantly lower than those in blank and negative control groups(both P 0.05).Conclusion Recombinant adenovirus Ad-U6-COX-2-shRNA-EGFP was constructed successfully,which inhibited the proliferation of SMMC-7721 cells significantly.The study solved the problems in clinical application and laid a foundation of research on therapy of liver cancer as well as the relevant molecular mechanism.